Mutations in the BCR-ABL tyrosine kinase associated with resistance to ST1-571

ABSTRACT

The invention described herein relates to novel genes and their encoded proteins, termed Mutants Associated with Resistance to STI-571 (e.g., T315I Bcr-Abl), and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express MARS. The invention further provides methods for identifying molecules that bind to and/or modulate the functional activity of MARS.

RELATED APPLICATIONS

This application claims priority under Section 119(e) from U.S.Provisional Application Ser. No. 60/298,728 filed Jun. 14, 2001 and U.S.Provisional Application Ser. No. 60/331,709 filed Nov. 20, 2001, thecontents of each of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support by a USPHS NationalResearch Service Award GM07185 (M.E.G.). The Government may have certainrights in this invention.

FIELD OF THE INVENTION

The invention described herein relates to novel genes and their encodedproteins, and to diagnostic and therapeutic methods and compositionsuseful in the management of cancers that express them.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise andare predicted to become the leading cause of death in the developedworld.

Cancers are characterized by multiple oncogenic events that collectivelycontribute to the phenotype of advanced stage disease. With the adventof new drugs that target specific molecular abnormalities, it isimportant to know whether the initial oncogenic event continues to playa functional role at later stages of tumor progression and at relapsewith the development of chemotherapy resistance. This question has beenaddressed in transgenic mice through regulated expression of the initialoncogene. In three models testing different oncogenes in differenttissues, the primary oncogene was required to maintain the tumorphenotype, despite the presence of numerous additional oncogene andtumor suppressor mutations (see, e.g. L. Chin et al., Nature 400, 468(1999); D. W. Felsher et al., Mol. Cell 4, 199 (1999); and C. S.Huettner et al., Nature Genet. 24, 57 (2000)). Recent clinical trials ofthe Abelson tyrosine kinase (Abl) inhibitor STI-571 in chronic myeloidleukemia (CML) allow this question to be addressed directly in humancancer (see, e.g. B. J. Druker et al., N. Engl. J. Med. 344, 1038(2001); and B. J. Druker et al., N. Engl. J. Med. 344, 1031 (2001)).

CML is a pluripotent hematopoietic stem cell disorder characterized bythe Philadelphia (Ph) chromosome translocation (see, e.g. C. L. Sawyers,N. Engl. J. Med. 340, 1330 (1999); and S. Faderl et al., N. Engl. J.Med. 341, 164 (1999)). The resulting BCR-ABL fusion gene encodes acytoplasmic protein with constitutive tyrosine kinase activity (see,e.g. J. B. Konopka et al., Proc. Natl. Acad. Sci. U.S.A. 82, 1810 (1985)and NCBI Accession NP_(—)067585). Numerous experimental models haveestablished that BCR-ABL is an oncogene and is sufficient to produceCML-like disease in mice (see, e.g. G. Q. Daley et al., Science 247, 824(1990); and N. Heisterkamp et al., Nature 344, 251 (1990)). CMLprogresses through distinct clinical stages. The earliest stage, termedchronic phase, is characterized by expansion of terminallydifferentiated neutrophils. Over several years the disease progresses toan acute phase termed blast crisis, characterized by maturation arrestwith excessive numbers of undifferentiated myeloid or lymphoidprogenitor cells. The BCR-ABL oncogene is expressed at all stages, butblast crisis is characterized by multiple additional genetic andmolecular changes.

A series of inhibitors, based on the 2-phenylaminopyrimidine class ofpharmacophores, has been identified that have exceptionally highaffinity and specificity for Abl (see, e.g., Zimmerman et al., Bloorg,Med. Chem. Lett. 7, 187 (1997). The most successful of these, STI-571(formerly referred to as Novartis test compound CGP 57148 and also knownas Gleevec and imatinib), has been successfully tested in clinical traila therapeutic agent for CML. STI-571 is a 2-phenylamino pyrimidine thattargets the ATP-binding site of the kinase domain of ABL (see, e.g. B.J. Druker et al., Nature Med. 2, 561 (1996)). In phase I clinicaltrials, STI-571 induced remissions in patients in chronic phase as wellas blast crisis (see, e.g. B. J. Druker et al., N. Engl. J. Med. 344,1038 (2001); and B. J. Druker et al., N. Engl. J. Med. 344, 1031(2001)). While responses in chronic phase have been durable, remissionsobserved in blast crisis patients have usually lasted only 2-6 months,despite continued drug treatment (see, e.g. B. J. Druker et al., N.Engl. J. Med. 344, 1038 (2001)).

In view of the relapse observed in patients treated with STI-571 thereis a need for an understanding of the mechanisms associated with STI-571resistance in CML and related cancers as well as diagnostic andtherapeutic procedures and compositions tailored to address thisphenomena. The invention provided herein satisfies this need.

SUMMARY OF THE INVENTION

Clinical studies with the Abl tyrosine kinase inhibitor STI-571 inchronic myeloid leukemia (CML) demonstrate that many patients withadvanced stage disease respond initially but then relapse. While,biochemical and molecular analysis of clinical materials from thesepatients shows that drug resistance is associated with reactivation ofBcr-Abl signal transduction, the specific events associated with thisresistance have not been not well characterized.

The disclosure provided herein characterizes specific events associatedwith such drug resistance by identifying specific domains within proteinkinases where amino acid mutations occur that impart resistance to thekinase inhibitor yet allow the kinase to retain its biological activity.The disclosure provided herein further identifies these regions asdomains shown to be highly conserved among families of protein kinases(e.g. the c-Abl tyrosine kinase activation loop). Consequently thisdisclosure identifies those specific regions in protein kinases that areto be analyzed in a variety of diagnostic protocols which examine drugresistance.

The invention described herein further includes novel genes and theirencoded proteins expressed in cancer cells that are associated withresistance to STI-571. Typically these STI-571 resistant genes and theirencoded proteins are mutants of Bcr-Abl an oncogene that is expressed inchronic myeloid leukemias. The invention described herein discloses anumber of Bcr-Abl Mutants Associated with Resistance to STI-571hereinafter these mutants are collectively described using the acronym“MARS”), as well as diagnostic and therapeutic methods and compositionsuseful in the management of cancers that express these mutants.

A typical example of a MARS is a Bcr-Abl mutant having a single aminoacid substitution in a Thr residue at position 315 of the Abl kinase(termed T315I Bcr-Abl). In clinical studies, patients exhibited STI-571resistance associated with this mutation at residue 315, a residue inthe Abl kinase domain known to form a critical hydrogen bond with thisdrug. Biochemical analyses of this mutant show that the Thr→Ile changeis sufficient to confer STI-571 resistance in a reconstitutionexperiment. Additional MARS are identified in Tables I provided below.The disclosure provided herein presents evidence that geneticallycomplex cancers retain dependence on an initial oncogenic events andprovides a strategy for identifying inhibitors of STI-571 resistance.The disclosure provided herein further provides for a variety ofdiagnostic methods for examining the characteristics of cancers such aschronic myeloid leukemia.

All prior knowledge of the Bcr-Abl tyrosine kinase is based on publishedsequence that has been in the public domain for >15 years. The inventionprovides novel sequences of DNA of the Bcr-Abl tyrosine kinase fusionprotein that causes chronic myeloid leukemia (CML), which is present ina high fraction of patients who develop resistance to the drug STI-571,which is soon to become standard of care for the treatment of CML. Asdisclosed herein, methods for evaluating the status of the MARSpolypeptides and polynucleotides it can be used in the evaluation ofcancers, for example to detect early relapse. Moreover, MARSpolypeptides and polynucleotides can be used to create assays toidentify drugs which inhibit the biological activity of these mutantproteins.

T315I Bcr-Abl provides a representative example of the inventionsprovided by the MARS disclosed herein. The T315I Bcr-Abl mutantdisclosed herein contains an amino acid change in the kinase domain ofBcr-Abl that inhibits STI571 binding to Bcr-Abl. The T315I Bcr-Ablembodiment of the invention has been tested in a number of patientsamples and confirmed at the sequence level. This mutant Bcr-Abl proteinhas been expressed in cells and shown to be resistant to STI-571.Therefore, patients develop resistance to the drug because it can nolonger inhibit its kinase activity.

Knowledge of mutant sequences provide immediate utility for a number ofmethods. In particular, currently there are no methods for detecting ortreating drug-resistant CML. Consequently, the invention provided hereinprovides diagnostic tests for early relapse in CML as well as for drugdevelopment in the field of tyrosine kinase inhibitors. For example, thedisclosure provided herein allows one to detect the presence of drugresistant cells in CML patients prior to relapse, using, for example,PCR based assays. Representative embodiments of the invention includePCR and analogous assays that are used to detect resistant cells inpatient blood samples.

The invention can also be practiced as a tool to identify moleculeswhich bind and/or inhibit the mutant tyrosine kinases. A typicalembodiment of this aspect of the invention is a method of identifying acompound which specifically binds to a mutant protein kinase such as aBcr-Abl mutant shown in Table I by contacting the mutant with a testcompound under conditions favorable to binding; and then determiningwhether said test compound binds to the mutant so that a compound whichbinds to the mutant is identified. Using such methods one can performstructure-based drug design and/or high throughput screening of chemicallibraries to identify inhibitors of mutant tyrosine kinases. Such aninhibitor will have immediate clinical relevance.

The invention provides polynucleotides corresponding or complementary toall or part of the MARS genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding MARSproteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to the MARSgenes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the MARS genes, mRNAs, or toMARS-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding MARS. Recombinant DNA molecules containingMARS polynucleotides, cells transformed or transduced with suchmolecules, and host-vector systems for the expression of MARS geneproducts are also provided. The invention further provides MARS proteinsand polypeptide fragments thereof. The invention further providesantibodies that bind to MARS proteins and polypeptide fragments thereof,including polyclonal and monoclonal antibodies, murine and othermammalian antibodies, chimeric antibodies, humanized and fully humanantibodies, and antibodies labeled with a detectable marker.

The invention further provides methods for detecting the presence andstatus of MARS polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express MARS. Atypical embodiment of this invention provides methods for monitoringMARS gene products in a tissue sample having or suspected of having someform of growth dysregulation such as cancer.

One preferred embodiment of the invention is a method of identifying amutant Abelson tyrosine kinase expressed by a cell by determining anucleotide sequence of a portion of the catalytic domain of the Abelsontyrosine kinase expressed by the cell and then comparing the nucleotidesequence so determined to that of the wild type sequence of thecatalytic domain of the Abelson protein tyrosine kinase to identify thepresence of a mutation within the catalytic domain, wherein the mutationso identified has the characteristics of occurring at a amino acidresidue located within the polypeptide sequence of the Abelson proteintyrosine kinase at a amino acid residue that has homology to an aminoacid position in a Bcr-Abl kinase shown in SEQ ID NO: 1 that isassociated with a resistance to an inhibition of tyrosine kinaseactivity by a 2-phenylaminopyrimidine, wherein the homology between theamino acid residue located within the polypeptide sequence of theAbelson protein tyrosine kinase and the amino acid residue in theBcr-Abl kinase shown in SEQ ID NO: 1 that is associated with aresistance to an inhibition of tyrosine kinase activity by a2-phenylaminopyrimidine can be illustrated via a BLAST analysis.

Another embodiment of the invention is an isolated Bcr-Abl polypeptidecomprising an amino acid sequence which differs from the sequence of theBcr-Abl of SEQ ID NO:1 and has one or more amino acid substitutions atthe residue position(s) in SEQ ID NO:1 selected from the groupconsisting of: D233, T243, M244, K245, G249, G250, G251, Q252, Y253,E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274,D276, T277, M278, E282, F283, A288, M290, K291, E292, I293, P296, L298,V299, Q300, G303, V304, C305, T306, F311, I314, T315, E316, F317, M318,Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342,M343, A344, I347, A350, M351, E352, E355, K357, N358, F359, I360, L364,E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396,A399, P402, and T406. A related embodiment of the invention is anisolated nucleic acid comprising a nucleotide sequence encoding theBcr-Abl polypeptide. Other embodiments of the invention is a vectorcomprising this nucleic acid sequence, a host cell comprising suchvectors (e.g. E. coli) as well as a method of making Bcr-Abl polypeptidevariant polypeptide, comprising the steps of: providing a host cellcomprising such a vector; (b) providing culture media; (c) culturing thehost cell in the culture media under conditions sufficient to expressthe Bcr-Abl polypeptide variant polypeptide; (d) recovering the Bcr-Ablpolypeptide variant polypeptide from the host cell or culture media; and(e) purifying the Bcr-Abl polypeptide variant polypeptide. Yet anotherembodiment of the invention is a Bcr-Abl polypeptide variant polypeptidethat is chemically modified or conjugated or linked to a matrix or aheterologous protein.

The invention further provides various therapeutic compositions andstrategies for treating cancers that express MARS, including methods foridentifying molecules (e.g. STI-571 analogs) which inhibit thebiological activities (e.g. kinase activity) of various MARS.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Clinical relapse of STI-571-treated patients is associated withpersistent Bcr-Abl kinase activity. (A) Immunoblot analyses of one CMLpatient's bone marrow cells after a 2-hour incubation with differentconcentrations of STI-571 in vitro. Whole cell lysates were separated bySDS-PAGE, transferred to nitrocellulose, and probed with Crkl (toppanel), phosphotyrosine (middle panel), and Abl (bottom panel)antibodies. (B) Crkl immunoblot of whole cell lysates from CML patientsprior to STI-571 therapy (left) and from Ph-positive blast crisispatients who achieved hematological remission (<5% blasts) on STI-571but remained 100% BCR-ABL-positive (right). (C) Crkl immunoblots ofwhole cell lysates from lymphoid blast crisis or Ph-positive acutelymphoid leukemia patients (top panel) and myeloid blast crisis patients(middle panel) who relapsed after initially responding to STI-571therapy. Phosphotyrosine immunoblot of patient cell lysates at time ofrelapse (bottom panel). Ph-positive cell line, K562, was used as apositive control for autophosphorylated Bcr-AbL (D) Crkl immunoblots ofcell lysates from relapse patients taken prior to (pre-Tx) and duringthe course of (Tx and relapse) STI-571 therapy. Densitometric analysesof Crkl immunoblots (expressed as percentage of phosphorylated Crkl overtotal Crkl protein) are presented in bar graphs.

FIG. 2. Altered sensitivity of relapsed patient cells to STI-571. (A)STI-571 dose-response curves of Crkl phosphorylation in cells taken fromblast crisis patients (LB3 and LB2) prior to STI-571 therapy (◯) and atthe time of relapse (). Cells from both time points were exposed toincreasing concentrations of STI-571, harvested, and analyzed by Crklimmunoblot and densitometry. (B) IC₅₀ values for inhibition of Crklphosphorylation determined by exposure of cells isolated from untreatedversus relapsed CML patients to increasing concentrations of STI-571,and subsequent Crkl immunoblot and densitometric analyses. Crklphosphorylation in one relapsed patient sample (LB2) could not beinhibited with high concentrations of STI-571. (IC₅₀=concentration ofSTI-571 required to reduce CRKL phosphorylation by 500%).

FIG. 3. BCR-ABL amplification in patients who relapsed after an initialresponse to STI-571. (A) BCR-ABL FISH analyses of interphase nuclei fromblast crisis patient M13 prior to and during STI-571 therapy. Nuclei arevisualized with DAPI stain (blue), ABL probe is labeled with SpectrumOrange (red signal) and BCR probe is labeled with Spectrum Green (greensignal). BCR-ABL gene fusions, indicated by yellow signals, show anincrease in BCR-ABL gene amplification during STI-571-resistant diseaseprogression. (Bar=10μ. (B) BCR-ABL FISH analyses of interphase nucleifrom blast crisis patient M14 prior to, during, and after removal fromSTI-571 therapy showing BCR-ABL-amplified phenotype and reversion tonon-amplified phenotype upon removal from STI-571 therapy. (Bar=10μ. (C)Giemsa stained image (left panel; Bar=5μ) and dual color FISH images(middle and right panels; Bars=3μ of sample from patient LB1 showingduplicated inverted Ph-chromosome. Arrows indicate BCR-ABL gene fusions.(D) Quantitative PCR analysis of genomic DNA from BCR-ABL-amplifiedpatients (MB13, MB14, LB1) and one non-amplified patient LB3 (control)confirming increased ABL gene copy number in all three patients.

FIG. 4. Point mutation in the ATP-binding pocket of the Abl kinasedomain confers STI-571 resistance in relapsed patients. (A) Schematic ofPCR strategy to determine the sequence of a 578 base pair region ofBCR-ABL that corresponds to the ATP-binding pocket and activation loopof the kinase domain in patient samples. Amino acid sequence of theregion of Abl analyzed is shown in black. Residues predicted to formhydrogen bonds with STI-571, based on crystal structure data, are inboldface and are numbered from the first amino acid of c-Abl (GenBankaccession number: M14752, shown in Table II) (SEQ ID NO: 1).Corresponding nucleotide sequence (shown in red) was aligned withsequences obtained from nine patient cDNAs. The C→T mutation at ABLnucleotide 944 (detected in six patients at relapse and in nopre-treatment samples) is shown in blue. Sequence of wild-type ABL exon3 (GenBank accession number: NT008338.2) was aligned with sequencesobtained from patient genomic DNA prior to treatment and at relapse.Examples of primary sequence data (represented as chromatographs) fromwild-type BCR-ABL (left) and BCR-ABL with the C→T point mutation(right). (B) Model of STI-571-binding pocket of wild-type Abl in complexwith STI-571 (left panel) and predicted structure of STI-571-bindingpocket of T315I mutant Abl in complex with STI-571 (right panel). In themolecular structures representing STI-571 and Abl residue 315, nitrogenatoms are shown in blue and oxygen atoms are shown in red. Van-der-Waalsinteractions are depicted in grey for STI-571 (both panels), in blue forwild-type Abl residue Thr³¹⁵ (left panel), and in red for mutant Ablresidue Ile³¹⁵ (right panel). Polypeptide backbone of the Abl kinasedomain is represented in green. (C) Immunoblots of whole cell lysatesisolated from transfected 293T cells (wild-type p210 BCR-ABL shown inleft panels and T315I mutant shown in right panels) after a 2-hourincubation with different concentrations of STI-571. Blots were probedwith phosphotyrosine (top panels) and Abl (bottom panels) antibodies.

FIG. 5. Graphic schematic of mutations in more than one patient.

FIG. 6. Bar graph schematic of total mutations in 2 or more patients.

FIG. 7. Geldanamycin and 17-AAG induce degradation of wild-type andSTI571-resistant, mutant BCR-ABL proteins and inhibit BCR-ABL signaling.(A) Ba/F3 cells expressing wild-type, T315I, or E255K BCR-ABL wereincubated in the presence of increasing concentrations of geldanamycin(GA) for 24 hours. Immunoblotting of cell lysates was performed withanti-ABL (Ab3, Oncogene) (upper panels), anti-RAF-1 (Santa Cruz) (middlepanels), and anti-actin (ac-15, Sigma) as a control for protein loading(lower panels). (B) Ba/F3 cells expressing wild-type, T315I, or E255KBCR-ABL were incubated in the presence of increasing concentrations of17-AAG for 24 hours. Immunoblotting of these lysates was performed withanti-ABL (upper panels) and anti-actin as a control for protein loading(lower panels). (C) Immunoblotting of the same lysates from (B) wasperformed with anti-CRKL (Santa Cruz). CRKL, whentyrosine-phosphorylated, migrates more slowly on SDS-PAGE resulting inan upper band representing phosphorylated CRKL (P-CRKL) and a lower bandrepresenting non-phosphorylated CRKL. (D) Densitometric analysis of CRKLimmunoblot shown in (C) using ImageQuant software (Molecular Dynamics).Quantified CRKL phosphorylation is expressed as percentage ofphosphorylated CRKL over total CRKL protein (% P-CRKL). (E)Densitometric analysis of CRKL immunoblotting using lysates from thesame Ba/F3 cell lines incubated in the presence of increasingconcentrations of STI571 for 24 hours.

FIG. 8. Schematic of Bcr-Abl kinase domain sequencing methodology.Bcr-Abl cDNA is represented with Bcr sequences stippled, and Ablsequences in black. Horizontal arrows represent PCR primers. Initial PCRresults in amplification of a 1.3 kb Bcr-Abl subfragment which serves astemplate for a second round PCR of the kinase domain which is thensubcloned. Ten independent clones per patient time point were sequenced.Sequence deviations from wild-type Bcr-Abl observed in at least two often clones were considered mutations.

FIG. 9. Bcr-Abl kinase domain mutants exhibit varying degrees ofbiochemical and biologic resistance to STI-571. Western blot using ananti-phosphotyrosine antibody (4G10) of lysates prepared from Ba/F3populations infected with retroviruses expressing the Bcr-Abl isoformsindicated and grown in the absence of IL-3 were exposed to varyingconcentrations of STI-571 for two hours are shown. Biochemical IC-50sfor each of the mutations is shown. Biologic IC-50s were determined byviable cell count of cells after 48 hours of STI-571 exposure.

FIG. 10. Imatinib-resistant mutations occur over a wide range of theBcr-Abl kinase domain. The kinase domain amino acid sequence ofwild-type Bcr-Abl is shown. Asterisks mark the conserved amino acids ofthe Gly-X-Gly-X-X-Gly-X-Val consensus sequence found within the P-loop.Amino acid substitutions found in STI-571-resistant patients areindicated beneath the wild-type sequence.

FIG. 11. Summary of STI-571-resistant Bcr-Abl kinase domain mutations.Each letter represents a patient with in whom the corresponding mutationwas detected. Chronic phase patients are represented by the letter “C.”Relapsed myeloid blast crisis patients are indicated by the letter “M.”Patients with relapsed lymphoid blast crisis are represented by theletter “L.” “R” indicates mutations prior to STI-571 treatment inpatients with myeloid blast crisis who were refractory to treatment.Note that kinase domain is not drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., 1989, Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,Current Protocols in Molecular Biology, Wiley Interscience Publishers,(1995). As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least about 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least about6 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than, for example, the MARS genes orthat encode polypeptides other than MARS gene product or fragmentsthereof. As used herein, a polypeptide is said to be “isolated” when itis substantially separated from contaminant polypeptide that correspondto polypeptides other than the MARS polypeptides or fragments thereof. Askilled artisan can readily employ polynucleotide or polypeptideisolation procedures to obtain an isolated polynucleotides andpolypeptides.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C., and most preferably tostringent hybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium. citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., 1989, Molecular Cloning: A Laboratory Manual New York:Cold Spring Harbor Press, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

For purposes of shorthand designation of BCR-ABL variants describedherein, it is noted that numbers refer to the amino acid residueposition along the amino acid sequence of the BCR-ABL polypeptide. Aminoacid identification uses the single-letter alphabet of amino acids,i.e.,

Asp D Aspartic acid Ile I Isoleucine Thr T Threonine Leu L Leucine Ser SSerine Tyr Y Tyrosine Glu E Glutamic acid Phe F Phenylalanine Pro PProline His H Histidine Gly G Glycine Lys K Lysine Ala A Alanine Arg RArginine Cys C Cysteine Trp W Tryptophan Val V Valine Gln Q GlutamineMet M Methionine ASN N Asparagine

In the context of amino acid sequence comparisons, the term “identity”is used to identify and express the percentage of amino acid residues atthe same relative positions that are the same. Also in this context, theterm “homology” is used to identify and express the percentage of aminoacid residues at the same relative positions that are either identicalor are similar, using the conserved amino acid criteria of BLASTanalysis, as is generally understood in the art. For example, identityand homology values may be generated by WU-BLAST-2 (Altschul et al.,Methods in Enzymology, 266: 460-480 (1996):http://blast.wustl/edu/blast/README.html).

“Percent (%) amino acid sequence identity” with respect to the sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe BCR-ABL sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art can determine appropriate parameters for measuring alignment,including assigning algorithms needed to achieve maximal alignment overthe full-length sequences being compared. For purposes herein, percentamino acid identity values can also be obtained using the sequencecomparison computer program, ALIGN-2, the source code of which has beenfiled with user documentation in the US Copyright Office, Washington,D.C., 20559, registered under the US Copyright Registration No.TXU510087. The ALIGN-2 program is publicly available through Genentech,Inc., South San Francisco, Calif. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particularexamples of such cancers include chronic myeloid leukemia, acutelymphoblastic leukemia, squamous cell carcinoma, small-cell lung cancer,non-small cell lung cancer, glioma, gastrointestinal cancer, renalcancer, ovarian cancer, liver cancer, colorectal cancer, endometrialcancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma,pancreatic cancer, glioblastoma multiforme, cervical cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, andhead and neck cancer.

The terms “treating”, “treatment” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy. Theterms “individual selected for treatment” refer to an individual who hasbeen identified as having a condition that artisans understand canrespond to a specific therapy and, consequentially is being consideredfor treatment (or being treated with) that therapy (e.g. an individualsuffering from chronic myelogenous leukemia who is being treated withSTI-571).

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

Additional definitions are provided throughout the subsections thatfollow.

The invention described herein relates to novel genes and their encodedproteins, termed Mutants Associated with Resistance to STI-571 (e.g.,T315I Bcr-Abl), and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that expressMARS. Embodiments of the invention provided herein are illustrated bystudies of the Bcr-Abl protein kinase in STI-571-treated patients. Tocharacterize the mechanism of relapse in STI-571-treated patients, wefirst assessed the status of Bcr-Abl signaling in primary leukemiacells. As discussed in the Examples below, peripheral blood and/or bonemarrow samples were obtained with appropriate informed consent from CMLand Ph-positive ALL patients at UCLA who were enrolled in multicenterclinical trials of STI-571 sponsored by Novartis Pharmaceuticals. Allpatients had >30 percent blasts in the marrow prior to treatment.Responding patients had reduction in the percentage of bone marrowblasts to <15 percent (partial) or <5 percent (complete), as describedin B. J. Druker et al., N. Engl. J. Med. 344, 1031 (2001). Progressivedisease was defined as an increase in percentage of blasts after aninitial response, despite continued STI-571 treatment. Mononuclear cellswere isolated by centrifugation through Ficoll-Hypaque, washed twice inphosphate-buffered saline, counted and used immediately orcryopreserved.

A goal was to distinguish between Bcr-Abl dependent versus Bcr-Ablindependent mechanisms of relapse. If Bcr-Abl remains critical forproliferation of the leukemia clone, then the Bcr-Abl signaling pathwayshould be reactivated. Alternatively, if expansion of the leukemia cloneis independent of Bcr-Abl, then signaling through the Bcr-Abl pathwayshould remain impaired by STI-571. The most direct measure of signalingthrough Bcr-Abl pathway is the enzymatic activity of Bcr-Abl proteinitself (see, e.g. J. B. Konopka et al., Proc. Natl. Acad. Sci. U.S.A.82, 1810 (1985); S. S. Clark et al., Science 235, 85 (1987); and S. S.Clark et al., Science 239, 775 (1988)).

Although the enzymatic activity of Bcr-Abl protein is readily measuredin cell lines, such assays are difficult to perform in a reproducible,quantitative fashion with clinical material because Bcr-Abl is subjectto rapid degradation and dephosphorylation upon cell lysis. In a searchfor alternative measures of Bcr-Abl kinase activity, we found that thephosphotyrosine content of Crkl, an adaptor protein which isspecifically and constitutively phosphorylated by Bcr-Abl in CML cells(see, e.g. J. ten Hoeve et al., Blood 84, 1731 (1994); T. Oda et al., J.Biol. Chem. 269, 22925 (1994); and G. L. Nichols et al., Blood 84, 2912(1994)), could be measured reproducibly and quantitatively in clinicalspecimens (see Example 2 below). Crkl binds Bcr-Abl directly and plays afunctional role in Bcr-Abl transformation by lining the kinase signal todownstream effector pathways (see, e.g. K. Senechal et al., J. Biol.Chem. 271, 23255 (1996)). When phosphorylated, Crkl migrates withaltered mobility in SDS-PAGE gels and can be quantified usingdensitometry. As expected, Crkl phosphorylation in primary CML patientcells was inhibited in a dose-dependent manner when exposed to STI-571and correlated with dephosphorylation of Bcr-Abl (FIG. 1A). This Crk1assay allows for an assessment of the enzymatic activity of Bcr-Ablprotein in a reproducible, quantitative fashion in clinical materials.

A recent preclinical study of STI-571 resistance in mice engrafted witha human blast crisis CML cell line demonstrated that al acidglycoprotein, an acute-phase reactant synthesized by the liver, can bindSTI-571 in serum and block its activity against Bcr-Abl (see, e.g. C.Gambacorti-Passerini et al., J. Natl. Cancer Inst. 92, 1641 (2000)).This observation raises the possibility that STI-571 resistance inpatients is due to a host-mediated response against the drug.Alternatively, resistance might be mediated by a cell-autonomous eventin a leukemia subclone that allows escape from kinase inhibition bySTI-571. To distinguish between these two possibilities, we determinedthe sensitivity of patient cells taken prior to treatment and at thetime of relapse to STI-571 by measuring inhibition of Crklphosphorylation. Briefly, purified cells were plated at 1−10×10⁶/ml inRPMI-1640+10% human AB serum with varying concentrations of STI-571 for24 hours. Proteins were extracted and subjected to immunoblot analysis.

If STI-571 resistance is a consequence of a host response, pretreatmentand relapse leukemia cells should be equally sensitive to ex vivoSTI-571 treatment. However, if STI-571 is cell-intrinsic, leukemia cellsobtained at relapse should be less sensitive to STI-571 thanpretreatment cells. In those patients for whom we had sufficient matchedclinical material, a 10-fold or greater shift in sensitivity to STI-571was observed at relapse (FIG. 2A). Aggregate analysis of 11 samplesconfirmed that higher concentrations of STI-571 are required to inhibitCrkl phosphorylation in patients cells obtained at relapse versuspre-treatment (FIG. 2B).

Since these ex vivo studies provide evidence that STI-571 resistance iscell-intrinsic, we considered several possible mechanisms. Some CML celllines that develop resistance to STI-571 after months of in vitro growthin sub-therapeutic doses of the drug have amplification of the BCR-ABLgene (see, e.g. E. Weisberg et al., Blood 95, 3498 (2000); P. le Coutreet al., Blood 95, 1758 (2000); and F. X. Mahon et al., Blood 96, 1070(2000)). We performed dual-color fluorescence in situ hybridization(FISH) experiments to show that BCR-ABL gene amplification is similarlyimplicated in STI-571 resistance in human clinical samples (see Example3 below).

Through the disclosure, data from various groups of patients isdiscussed. Tables IA-IF provide a summary of patient data. Asillustrated in Example 1 below, we also considered the possibility thatmutations in BCR-ABL might confer resistance to STI-571. Consequently, a579 base pair region corresponding to the ATP-binding pocket and theactivation loop of the kinase domain of Bcr-Abl was sequenced in the 9patients for whom RNA was available at the time of relapse (FIG. 4A). Asingle, identical C→T nucleotide change was detected at ABL nucleotide944 in six of nine cases examined (FIG. 4A). In all six patients amixture of wild-type and mutant cDNA clones were found, with thefrequency of mutant clones ranging from 17% to 70%. The mutation wasfound in three of three patients with lymphoid disease and in three ofsix patients with myeloid blast crisis. The presence of the mutation wasconfirmed by analysis of genomic DNA (FIG. 4A).

In the MARS designated T315I Bcr-Abl, a single nucleotide C→T changeresults in a threonine to isoleucine substitution at position 315 ofc-Abl. The recently-solved crystal structure of the catalytic domain ofAbl complexed with a variant of STI-571 identified the amino acidresidues within the ATP-binding site and activation loop of c-Abl thatare required for STI-571 binding and thus inhibition of Abl kinaseactivity (see, e.g. T. Schindler et al., Science 289, 1938 (2000)).Thr³¹⁵ is among those that form critical hydrogen bonds with STI-571.The potential consequence of the T315I substitution on the STI-571binding pocket was modeled based on the crystal structure of thewild-type Abl kinase domain in complex with STI-571 (FIG. 4B). Theabsence of the oxygen atom normally provided by the side chain of Thr³¹⁵would preclude formation of a hydrogen bond with the secondary aminogroup of STI-571. In addition, isoleucine contains an extra hydrocarbongroup in the side chain, which would result in steric clash with STI-571and presumably inhibit binding. Notably, the model predicts that theT315I mutation should not interfere with ATP binding. The structure ofthe kinase domain of Hck in complex with an ATP analog (AMP-PNP) wassuperimposed onto the model of the Ile315 Abl kinase domain.

T315I Bcr-Abl is discussed as a representative embodiment of the MARSdisclosed herein (e.g. those described in Table IA below). In certaindescriptions of the invention provided herein, embodiments of a singlegene are used (T315I Bcr-Abl, for example) to illustrate typicalembodiments of the invention that apply to all of the MARS disclosedherein (e.g. E255K, Q252H, V304D, M351T, E355G etc. as shown in Table I)In this context, artisans understand that discussing a typicalembodiment directed to a single species (e.g. T315I) when theembodiments are commonly applicable to the other species disclosedherein (e.g. E255K, Q252H, V304D, M351T, E355G etc.) eliminatesunnecessary redundancy in the descriptions of the invention.

The T315I mutation is shown to preserve kinase activity and, based uponthe crystal structure of the kinase domain when bound to STI-571, ispredicted to result in ineffective binding of STI-571 to BCR/ABL. In aneffort to define the full spectrum of kinase domain mutations in alarger sample size, we sequenced the BCR/ABL kinase domain in 18patients with CML in myeloid blast crisis. In 13 patients, samplesobtained at the time of relapse after a partial or complete response toSTI-571 (acquired resistance) were analyzed. In 5 patients who did notrespond to STI-571 (de novo resistance), analysis was performed onsamples obtained prior to treatment. To ensure detection of subcloneswith kinase domain mutations that might account for a minority ofBCR/ABL expressing cells in the blood, we typically sequenced tenindependent clones from each patient sample. A mutation was consideredpresent only if it was detected by sequencing of both cDNA strands. Thepreviously identified T315I mutation was found in 3 additional patients.

In conjunction with our preliminary analysis of 11 patients (Gorre etal., 2001, Science, August 3; 293(5531):876-80), the T315I mutation hasalso been detected in subsequent studies of 9 of 28 patients (6/25myeloid blast crisis, 3/3 with lymphoid blast crisis or Ph+ ALL). Twoother mutations, M351T and E255K, were also found in 4 patients and 3patients respectively. Additional mutations were also found but did notalways represent the dominant subclone at time of relapse. Thesefindings indicate that BCR/ABL kinase domain mutations occur commonly inCML blast crisis and can be detected, in some cases, prior to STI-571treatment. These mutations may be a reflection of genetic instabilityassociated with disease progression or, possibly, prior treatmentexposure. Following the protocols used to examine the T315I mutation,which we have previously shown to cause in term resistance to STI-571,the significance of these additional mutations in STI-571 drugresistance can be defined.

To confirm that this amino acid substitution interferes with STI-571activity, we engineered the T315I mutation into wild-type p210 Bcr-Abl(see, e.g. Full-length p210 Bcr-Abl was subcloned into the pSRaMSVtkNeoretrovirus vector (see, e.g. A. J. Muller et al., Mol. Cell Biol. 11,1785 (1991)). A fragment containing the C to T mutation at ABLnucleotide 944 was made by PCR and swapped with the correspondingsequence in pSRaMSVtkNeo p210 Bcr-Abl wild-type to create thepSRaMSVtkNeo p210 Bcr-Abl T315I mutant. The resulting construct wasconfirmed by sequencing. Cells were transfected with wild-type or T315Ip210 Bcr-Abl and cultured in the presence of increasing concentrationsof STI-571. Briefly, the transient transfection of 293T cells wasperformed using CaCl₂ (see, e.g. A. J. Muller et al., Mol. Cell. Biol.11, 1785 (1991)). After a 24-hour transfection, cells were incubatedwith varying concentrations of STI-571 (provided by NovartisPharmaceuticals, Basel, Switzerland) for 2 hours. Proteins wereextracted and subjected to immunoblot analysis. As shown by Ablimmunoblot analysis, the expression of wild-type and T315I mutantBcr-Abl proteins was similar, and was not changed by STI-571 (FIG. 4C,bottom panels). Based on anti-phosphotyrosine immunoblot analysis, thekinase activities of wild-type Bcr-Abl and the T315I mutant appearcomparable in the absence of STI-571. Whereas wild-type Bcr-Abl kinaseactivity was inhibited by STI-571, the T315I mutant retained high levelsof phosphotyrosine at all concentrations of inhibitor tested (FIG. 4C,top panels).

In summary, our preliminary analysis of 11 patients with advanced stageCML who underwent disease progression after an initial response toSTI-571 shows that reactivation of Bcr-Abl signaling occurred in allpatients, despite continued STI-571 treatment. Therefore, the primaryexplanation for disease progression in these patients appears to beBcr-Abl dependent proliferation rather than secondary oncogenic signalsthat permit Bcr-Abl independent growth. It is possible that studies of alarger number of patients may identify exceptions to this theme, as hasbeen reported in transgenic mice expressing conditional oncogenes wherean occasional tumor can escape dependence on the initiating oncogene(see, e.g. L. Chin et al., Nature 400, 468 (1999); D. W. Felsher et al.,Mol. Cell. 4, 199 (1999); and C. S. Huettner et al., Nature Genet. 24,57 (2000)). In the majority of patients we studied, the mechanism ofresistance is a consequence of mutation or amplification of the targetoncogene BCR-ABL (one patient had both events). These results provideevidence in a genetically complex human cancer that a single moleculartarget remains relevant in late stage, relapsed disease.

Interestingly for example, the identity of the Abl kinase domainmutation found in these patients bears remarkable similarity to athreonine to isoleucine change in v-Src versus c-Src at position 338,which corresponds to Thr³¹⁵ in c-Abl. Despite the fact that v-Src andc-Src have almost identical kinase domain sequences (98% identity),v-Src is approximately 50-fold more resistant than c-Src to kinaseinhibition by the Src inhibitor PP1 (see, e.g. Y. Liu et. al., Chem.Biol. 6, 671 (1999).

Eleven Patients described in our preliminary study obtained completehematologic remissions and, in some cases, complete cytogeneticremissions on STI-571, then relapsed within two to six months. Thisclinical scenario must be distinguished from those of patients whoobtain only partial responses to STI-571 or fail to respond at all. In aphase II trial of 260 patients treated with STI-571 in myeloid blastcrisis, only about 20% of patients fell into the former group.Therefore, the 11 patients described in our study represented a highlyselect population. This distinction is important, because patients withpartial hematologic responses and no cytogenetic response will have asubstantial number of mature BCR-ABL expressing hematopoietic cells thatpersist during treatment and are not representative of the relapsing,drug-resistant subclone. Since the current protocols for mutationdetection do not specifically isolate relapsing, drug-resistant cellsfrom other BCR-ABL ex-pressing blood cells, failure to detect a mutationmight be explained by an insensitive assay. In contrast, the dominantpopulation of BCR-ABL expressing cells in patients who relapse after acytogenetic response will, by definition, be representative of theresistant subclone. Indeed, we found the T315I mutation in more than 80%of BCR-ABL expressing cells from three such patients.

With respect to methodology, we subcloned our PCR products rather thanperform direct sequencing, and we sequenced at least 10 independentclones per patient. All mutations required confirmation by sequencing inboth directions. We chose this strategy to maximize our sensitivity ofdetecting mutations that may be present in a minority ofBCR-ABL-expressing cells. In addition, this method provided a roughquantitative estimate of the fraction of BCR-ABL-expressing cells thatcontained the mutation, so that clonal evolution could be monitored overtime. In retrospect, this method allowed us to find the T315I mutationin several patients in whom the resistant clone represented less than20% of the BCR-ABL-expressing cells.

Although the development of STI-571 resistance presents new therapeuticchallenges, the fact that Bcr-Abl remains active in STI-571-resistantcells provides evidence that the chimeric oncoprotein remains a rationaldrug target. Because a significant fraction of the patients examined todate share an identical mutation associated with drug resistance, it maybe possible to identify an inhibitor of the mutant BCR-ABL allele thatwould have broad utility. In addition, knowledge of this mutationprovides for the development of a wide variety of assays to evaluatethis mutation, for example to detect drug resistant clones prior toclinical relapse. See, e.g. B. J. Druker et al., N. Engl. J. Med. 344,1038 (2001); A. Goga et al., Cell 82, 981 (1995); E. Abruzzese et al.,Cancer Genet. Cytogenet. 105, 164 (1998); J. D. Thompson et al.,Nucleic. Acids Res. 25, 4876 (1997); A. J. Muller et al., Mol. Cell.Biol. 11, 1785 (1991).

As noted herein, analysis has revealed that STI-571 resistance can occurthrough at least two distinct mechanisms. Some patients developchromosomal amplification of the genomic region encoding Bcr-Abl,resulting presumably in levels of Bcr-Abl protein that overcome theintracellular concentration of STI-571 (Gorre et al., Science293:876-880 (2001)). A second mechanism involves point mutations in thekinase domain that presumably interfere with drug-protein bindingwithout compromising kinase activity. The best characterized of theseinvolves a substitution of isoleucine for threonine at amino acidposition 315 (T315I) which alters the shape of the drug-binding pocketbased on a crystallographic-based model (Gorre et al., Science293:876-880 (2001)). A limited number of other mutations within thekinase domain has been reported at frequencies ranging from two of 44cases (Barthe et al., Science 293:2163 (2001); Hochhaus et al., Science293:2163 (2001)) to seven of eight in cases of acquired resistance (VonBubnoff et al., Lancet 359:487-491 (2001); Lancet 359:487-491 (2001)).The result of one small study revealed no detectable mutations in asingle patient with accelerated phase CML at the time of relapse, butfound unique mutations, including E255K in each of five patients withPhiladelphia chromosome-positive acute lymphoblastic leukemia (ALL), aswell as in one patient with CML in lymphoid blast crisis (Von Bubnoff etal., Lancet 359:487-491 (2001)). A separate study also focused uponPhiladelphia chromosome-positive ALL and detected E255K in six of ninepatients at the time of relapse, as well as T315I in one of ninepatients (Hofmann et al., Blood 99:1860-1862 (2002)). Most recently,results from a heterogenous group of patients revealed the presence ofBcr-Abl kinase domain mutations in two of four cases of relapsed myeloidblast crisis (one patient was found to harbor T315I, and the secondrevealed evidence of a novel mutation, G250E), and in two of sevenpatients with chronic phase disease who suffered progressive diseaseafter initial hematologic response. Evidence of new mutations G250E,F317L, and M351T was presented, but no biologic or biochemical assayswere reported (Branford et al., Blood 99:3472-3475 (2002)).

Given the reliance of leukemic cells upon Bcr-Abl activity at the timeof STI-571 resistance, efforts to overcome STI-571 resistance must beequipped to deal with the most common mechanisms of resistance. Otherinvestigators have reported widely varying frequencies of kinase domainmutations using methodology that involved direct sequencing of cDNA,which represents a consensus of sequences presence at the time ofrelapse. We sought to define the full spectrum of Bcr-Abl kinase domainmutations in cases of resistance using methods of mutation detectionwith superior sensitivity. Here we report our sequence analyses of theBcr-Abl kinase domain in patients treated with STI-571. These includecases of acquired resistance of myeloid blast crisis phase, cases ofmyeloid blast crisis exhibiting de novo resistance, cases of lymphoidblast crisis, and cases of chronic phase cytogeneticrefractoriness/relapse. We found evidence of Bcr-Abl kinase domainmutations in nearly all cases of acquired resistance.

Analysis of a subgroup of the more common mutations provides evidencethat these mutant isoforms retain the biologic activity of Bcr-Abl butexhibit varying degrees of resistance to STI-571 in both biochemical andbiological assays. Kinase domain point mutations apparently represent acommon mechanism through which resistance to STI-571 is acquired.Additionally, we provide the first evidence of polyclonal resistance toSTI-571 in individual patients. Efforts to target Bcr-Abl in the settingof STI-571-resistance will need to address the activities of thenumerous mutant Bcr-Abl isoforms. Medical management of CML patientsreceiving targeted therapy will likely be facilitated by routineperiodic assessment for kinase domain mutations. Lastly, we provideevidence for pre-existing kinase domain mutations in a small number ofpatients with STI-571-refractory myeloid blast crisis prior toinstitution of therapy, suggesting that the evolution of chronic phaseCML to blast crisis CML may be, in some cases, facilitated by theaccumulation of activating Bcr-Abl kinase domain mutations. Humanmalignancies, even those believed to rely upon a very small number ofgenetic alterations, likely comprise a significantly heterogeneouspopulation of cells, and the developing field of targeted therapy ofmalignancy appears to face daunting obstacles.

In an effort to define the true incidence and full spectrum of kinasedomain mutations that are capable of causing resistance in cases ofmyeloid blast crisis, we performed sensitive sequence analysis of theBcr-Abl kinase domain in patients whose disease relapsed after aninitial response to STI-571 treatment (“acquired resistance”). Weidentified different mutations in patients with relapsed myeloid blastcrisis in the vast majority of cases evaluated. Evidence of mutation wasfound in all four of the variable P-loop consensus(Gly-X-Gly-X-X-Gly-X-Val) amino acids. Mutations were found as far as140 amino acids away from the P-loop, and could be grouped by locationinto categories. Moreover, we provide evidence that resistancefrequently involves a polyclonal expansion of Bcr-Abl expressing cells.

In vitro analysis of a subset of kinase domain mutations demonstratedvarying degrees of STI-571 resistance relative to wild-type Bcr-Abl. Wealso analyzed the Bcr-Abl kinase domain in patients with chronic phaseCML who had no cytogenetic response to STI-571 and found evidence ofkinase domain mutations in a number of cases analyzed. A subset of thesekinase domain mutations were identical to those seen in relapsed myeloidblast crisis cases. Significantly, the presence of kinase domainmutation in this setting strongly correlated with disease progressionand decreased overall survival. Lastly, we found evidence ofSTI-571-resistant kinase domain mutations prior to STI-571 treatment ina subset of patients with myeloid blast crisis who subsequently failedto respond to STI-571.

Multiple mutations in the Bcr-Abl kinase domain can be detected at thetime of resistance in cases of myeloid blast crisis. Cytogeneticanalysis of patients with myeloid blast crisis whose disease initiallyresponded to STI-571 revealed persistence of the Philadelphia chromosomein nearly 100 percent. We envisioned the possibility of resistant clonesemerging from this population of Bcr-Abl containing cells, and thereforelikely comprising a subset of the Philadelphia chromosome-containingcells at the time of relapse. By sequencing ten independent PCR productsper patient sample and requiring two independent isolates of a givenmutation, we detect mutations that comprise as few as twenty percent ofthe population of Bcr-Abl sequences. Sequence analysis of the Bcr-Ablkinase domain revealed evidence of point mutations in sixteen ofseventeen cases of relapsed myeloid blast crisis (MBC) CML at the timeof relapse (see Table IV). The previously identified T315I mutation wasdetected. E255K, which has been previously described (Von Bubnoff etal., Lancet 359:487-491 (2001); Hofmann et al., Blood 99:1860-1862(2002); Branford et al., Blood 99:3472-3475 (2002)), was also detected.M351T was also recently reported (Branford et al., Blood 99:3472-3475(2002)) and was also detected. The novel mutation Q252H as well as therecently reported G250E (Branford et al., Blood 99:3472-3475 (2002))were found at the time of relapse. Patients had one of two alternativesubstitutions at position Y253; including mutations which substitutedhistidine for tyrosine (Y253H), as previously described (Von Bubnoff etal., Lancet 359:487-491 (2001); Branford et al., Blood 99:3472-3475(2002)). Interestingly, a novel conversion of tyrosine to phenylalanine(Y253F) was also observed. Phenylalanine is highly conserved at thisposition in the Src-family of tyrosine kinases, and when engineered into c-Abl, this mutation has been demonstrated to impart oncogenicity asreflected by cellular transformation assays (Allen et al., J Biol Chem275:19585-19591 (1996)). We have recently found the sensitivity of Y253Fto STI-571 to be intermediate between wild-type Bcr-Abl and the T315Imutant. Patients exhibited mutations within the activation loop atposition H396, involving substitution to either proline or arginine.Interestingly, several tyrosine kinases, including Hck, c-Src, v-src,lck, and Fyn all have an arginine at this position. Unique examples ofthe novel mutations V304D, E355G, and F359V, as well as the recentlyreported F317L (Branford et al., Blood 99:3472-3475 (2002)) were eachobserved. Using our method of detection analysis, we were able to detectBcr-Abl kinase domain mutations in the vast majority of samples obtainedfrom patients with relapsed myeloid blast crisis, including a number ofnovel mutations.

In addition to offering greater sensitivity of mutation detection, ourmethodology afforded the ability to assess for polyclonal resistance toSTI-571, i.e. the presence of more than one resistant clone in a givenpatient. Indeed, a significant percentage of patients with myeloid blastcrisis exhibiting acquired resistance to STI-571 were found to harbormore than one independent mutation. Samples obtained prior to treatmentin cases of acquired resistance exhibited no evidence of mutation.

To address whether the surprisingly high frequency and variety of kinasedomain mutations represented artifact introduced during the PCRamplification process, we sequenced ten independent subclones of the Ablkinase domain obtained from each of two healthy blood donors. Using ourcriteria of at least two independent isolates out of ten clones, wefound no evidence of Abl kinase domain mutation. We therefore concludethat the Bcr-Abl kinase domain mutations described here are highlyunlikely to be the result of PCR-introduced error, and most probablyrepresent accurate reflections of kinase domain sequence heterogeneityin these STI-571-resistant patients.

Imatinib-resistant cases of lymphoid blast crisis reveal kinase domainmutations similar to myeloid blast crisis. Analysis of samples obtainedfrom four of five patients with lymphoid blast crisis (LBC) at the timeof relapse revealed the presence of Bcr-Abl kinase domain mutations.Again, clear evidence for polyclonal resistance was observed, with thecoexistence of four separate mutations (Y253F, E255K, T315I, and M351T)in a single patient. Another patient harbored both E255K and Y253F. Twoadditional patients were found to harbor T315I in the absence of anyother mutations.

Bcr-Abl kinase domain mutations can be detected in chronic phasepatients who fail to achieve cytogenetic remission or lose anestablished major cytogenetic response and are associated with diseaseprogression and decreased survival. Cells from chronic phase patientswho failed to obtain cytogenetic remission or who lost a previouslyachieved cytogenetic remission were subjected to sequence analysis ofthe Bcr-Abl kinase domain. Analysis was performed on samples obtained atthe time of sustained hematologic response. A number of patients werefound to harbor mutations. Three of these mutations were also observedin cases of relapsed myeloid blast crisis described above (E255K, F317L,F359V). F317L was recently described in a single patient (Branford etal., Blood 99:3472-3475 (2002)) with chronic phase disease andcytogenetic persistence who subsequently suffered progressive disease.The last mutation, V379I, has not been documented in any other patientto date. Of the patients we studied, four have suffered progressivedisease and have since discontinued STI-571. Among these, three havedied and the fourth is living following subsequent allogeneic stem celltransplantation. Three of these four patients had Bcr-Abl kinase domainmutations (E255K, F317L, F359V) while one had no evidence of mutation.The patient harboring the V379I mutation continues to have a completehematologic remission in response to STI-571 in the absence of acytogenetic response. We conclude that kinase domain mutations occur inchronic phase patients who lose cytogenetic or hematologic responses toSTI-571, and in a subset of chronic phase patients who have persistenceof the Philadelphia chromosome in the setting of complete hematologicresponse.

Bcr-Abl kinase domain mutations can be detected prior to STI-571treatment in patients with myeloid blast crisis that exhibit de novoresistance, but not in patients with STI-571-sensitive myeloid blastcrisis or chronic phase CML. To determine whether Bcr-Abl kinase domainmutations may play a role in de novo resistance to STI-571, we analyzedpre-treatment samples from four patients with MBC who failed to achieveeven a transient response to STI-571. One patient exhibited T315I priorto initiation of therapy. Also detected in the same patient was aBcr-Abl allele that contained two mutations, M343T and F382L. A secondpatient had the E255K mutation prior to STI-571 treatment.

Bcr-Abl kinase domain mutations retain catalytic activity, and arecapable of conferring STI-571 resistance in vitro. To assess whether thenovel mutations observed were capable of conferring resistance toSTI-571 in vitro, we performed site-directed mutagenesis of Bcr-Abl in aretroviral expression plasmid. In an illustrative embodiment of theinvention, eight of the observed mutations (G250E, Q252H, Y253F, E255K,T315I, F317L, M351T, and E355G) were independently introduced intopSRalphaP210Bcr-Abl. While these mutants are provided as preferredembodiments of the invention described herein, those skilled in the artcan generate comparable mutants of any one of the MARS described hereinsuch as those identified in Table I.

Successful introduction of the expected mutations was confirmed bysequence analysis of the kinase domain. The eight mutations were eachtransiently transfected into 293-T cells, and found to exhibit varyingdegrees of sensitivity to STI-571, with IC-50 for enzymatic inhibitionin cells ranging from 1.27 uM to 5.63 uM as documented byphosphotyrosine-containing Bcr-Abl (see FIG. 9). The murinehematopoietic cell line Ba/F3 requires exogenous IL-3 in the absence ofBcr-Abl. Stable Ba/F3 cell lines, capable of growing in the absence ofinterleukin-3, were derived for each of the eight mutant isoforms,demonstrating that each of the eight mutant isoforms retains biologicactivity in this assay. The effect of varying concentrations of STI-571on cellular viability after 48 hours was determined. Again, the eightmutant isoforms were found to exhibit varying degrees of sensitivity toSTI-571. Several of the mutants appeared to impart only moderateresistance, retaining sensitivity to concentrations of STI-571 which aretheoretically achievable in patients (see FIG. 9).

Analysis of cells containing kinase domain mutations reveals no evidenceof point mutation in Bcr-Abl immediately sequences 5′ to the kinasedomain or in the tyrosine kinase domain of c-Kit. Genomic instabilityduring advanced phase CML has been previously described. The highfrequency of kinase domain mutations observed in our study, in additionto the finding of subpopulations of different mutations in individualpatients, could theoretically be a reflection of a global decrease inDNA mismatch repair, or alternatively, may reflect a strong selectionfor these isoforms in the presence of STI-571. In an effort to addressthis issue, sequencing of a 700 bp fragment of Bcr-Abl immediately 5′ tothe kinase domain was performed in five patients in whom several kinasedomain mutations were detected. No evidence of additional mutation wasfound in these samples. We also assessed the kinase domain of therelated tyrosine kinase c-Kit, which resides on chromosome 4 andexhibits sensitivity to STI-571 at concentrations equivalent to Bcr-Abl,in the same group of five patients. No evidence of c-Kit kinase domainmutation was detected, arguing against the possibility of widespreadgenomic instability. We hypothesize that the increased genomicinstability associated with blast crisis may result in a low backgroundof Bcr-Abl sequence variants, and STI-571 strongly selects for theemergence of kinase-active STI-571-resistant Bcr-Abl isoforms.

From the disclosure provided herein we conclude that with use ofsensitive detection methods, Bcr-Abl kinase domain mutations can bedetected in nearly all patients with relapsed myeloid blast crisis; thatresistance frequently involves the coexistence of cell populationscontaining different kinase domain mutations; that Bcr-Abl kinase domainmutations exhibit a wide range of STI-571 resistance in vitro; thatkinase domain mutations occur in a subset of chronic phase CML patientswith persistence of the Philadelphia chromosome, and portend a poorprognosis; and that some STI-571-resistant kinase domain mutations canbe occasionally detected in advanced phase cases CML prior to STI-571treatment, and therefore may contribute to the leukemic drive in cellsthat harbor them. Bcr-Abl kinase domain mutations may thus contribute tothe natural progression of CML from chronic to advanced phases in somecases. Given our findings, we believe routine sensitive sequenceanalysis of the Bcr-Abl kinase domain in patients being treated withSTI-571 is warranted.

As noted above, the disclosure provided herein supports kinase domainmutation as the primary mechanism for STI-571 failure. Previous studiesof kinase domain mutations have been performed largely on isolated casesof Philadelphia chromosome-positive ALL and CML in lymphoid blastcrisis. Our finding of Bcr-Abl kinase domain mutations in nearly allcases of relapsed myeloid blast crisis was not expected based uponprevious reports. Because the complete cytogenetic remission rate islower in myeloid blast crisis patients treated with Gleevec, it ispossible that resistance to Gleevec in patients with lymphoid blastcrisis CML and Ph+ ALL more commonly represents greater genetichomogeneity. Less sensitive methods of mutation detection may thereforeadequately demonstrate the presence of nucleotide substitutions in thesecases, yet fail to reliably detect mutations in relapsed myeloid blastcrisis cases.

While some previous studies suggested a predominance of one to twodifferent kinase domain mutations in the majority of STI-571-resistantcases, our expanded analysis of the Bcr-Abl kinase domain in resistantcases reveals a large spectrum of such mutations (see FIG. 10).Inspection of the P-loop, which contains the consensus sequenceGly-X-Gly-X-X-Gly-X-Val, reveals the presence of STI-571-resistantmutations at each of the non-conserved amino acid sites. Moreover,kinase domain mutations are exceedingly common in cases of acquiredresistance.

The methodology utilized in the current study represents the onlytechnique by which the sequence of individual mRNA molecules can bedetermined. We demonstrate here that cells from patients with acquiredresistance to STI-571 frequently represent a polyclonal population, withdifferent cells containing different Bcr-Abl kinase domain mutations.Furthermore, this methodology affords increased sensitivity by enablingthe detection of mutant isoforms that comprise as little asapproximately 20 percent of the resistant population of cells. In manyof our examples of polyclonal resistance, direct sequencing would bepredicted to yield a consensus wild-type kinase domain sequence, due tolack of a clonally dominant clone. Moreover, we provide the first directevidence for the presence of two separate kinase domain mutations on asingle strand of DNA. The method of mutation detection employed here isthus expected to be superior to the method of direct cDNA sequencingutilized by other investigators, particularly in cases where emergingresistance is the result of polyclonal expansion.

We further document the first evidence of kinase domain mutations incases of myeloid blast crisis prior to treatment with STI-571. While itis formally possible that such mutants merely reflect genomicinstability, the finding of such mutants at a frequency of twentypercent is more suggestive of a significant clonal expansion of thesecells. It is possible that certain kinase domain mutations may confer agrowth advantage in affected cells. The viral oncogene v-abl is known tocontain point mutations in addition to alternative N-terminal codingsequences when compared with murine c-abl. In this study we detectedT315I prior to treatment in a patient whose disease subsequently failedto respond to STI-571. Interestingly, at the corresponding residue inthe src gene, v-src differs from its cellular counterpart bysubstitution of isoleucine for threonine. Given a complete lack ofkinase-domain mutations in pretreatment samples obtained from patientswho subsequently responded to STI-571, the presence of kinase domainmutations prior to treatment may represent a marker for refractorydisease, most likely related to increased genetic heterogeneity.

We detected kinase domain mutations in the majority of chronic phasepatients who subsequently suffered progressive disease, and in only oneof nine patients who have a continued hematologic response on STI-571despite the persistence of the Philadelphia chromosome. The ability todetect kinase domain mutations in this setting thus appears to serve asa strong predictor for the likelihood of hematologic relapse. Moreover,all three patients with disease progression had evidence of theirmutations prior to exhibiting clinical signs of progressive disease.Periodic mutation analysis in this setting may be warranted tofacilitate alternative therapies. The only example of a detectablemutation in chronic phase CML without disease progression consisted ofthe V379I mutation, which has not been detected in any other patients.Given the correlation between the kinase domain mutations which havebeen shown to be functionally active in this study and diseaseprogression in the chronic phase despite STI-571 therapy, it may beuseful to periodically perform kinase domain mutation analysis ofpatients on STI-571 who have any degree of persistence of thePhiladelphia chromosome in an effort to anticipate disease progressionand to facilitate the prompt institution of allogeneic transplantationor other treatment options.

Our finding of mutant P210 isoforms in the overwhelming majority ofpatients at the time of acquired resistance reinforces point mutation inthe Bcr-Abl kinase domain as a primary reason for STI-571 failure. Thefuture of targeted therapy for CML is thus dependent upon overcomingSTI-571 resistance mediated by Bcr-Abl kinase domain mutations. Thedifferential sensitivity of kinase domain mutant isoforms to STI-571deserves consideration. Given the sensitivity of some mutants, such asF317L, M351T, and E355G, to concentrations of STI-571 theoreticallyobtainable in humans, trials of higher doses of STI-571 may be warrantedin some cases of acquired resistance. However, a few mutations, such asT315I, E255K, and G250E, clearly confer resistance to very highconcentrations of STI-571. We speculate that medical management in thefuture of both chronic and advanced phase CML exhibiting acquiredresistance to STI-571 will necessitate mutation-specific PCR, anddepending upon the presence or absence of certain mutations, doseescalation can be attempted. Should a highly resistant mutant isoform,such as T315I, E255K, or G250E subsequently achieve clonal dominance,second generation drugs with activity against the most STI-571-resistantisoforms could then be employed.

The clinical applicability of highly sensitive methods for mutationdetection is most well-established in the treatment of humanimmunodeficiency virus (HIV), where, armed with a number of targetedtherapies, clinicians make treatment decisions periodically based uponthe spectrum of retroviral mutations detected in the blood of theirpatients. Occasionally, drug-resistant mutations significantly hamperthe ability of virus to replicate, and anti-retroviral agents arewithdrawn in an effort to allow re-establishment of wild-type HIV. Itwill be important to characterize the biochemical and biologicalactivity of each of the various mutant Bcr-Abl isoforms. If, incomparison with wild-type Bcr-Abl, some STI-571-resistant mutationsactually impart decreased growth promoting effects, intermittent STI-571therapy could be instituted in an effort to delay disease progressiontoward the blast crisis stage.

The development of STI-571 for the treatment of CML continues torepresent a major advance toward the future of targeted therapy forhuman malignancies. Our work clearly implicates the activity of Bcr-Ablas essential to the malignant clone in nearly all acquired resistancecases studied. Imatinib is used much more commonly to treat chronicphase CML. Here we have provided examples of kinase domain mutations infour of fourteen cases of cytogenetic persistence despite STI-571therapy. The presence of kinase domain mutation strongly correlated withsubsequent development of progressive disease and decreased overallsurvival. The activity of Bcr-Abl therefore remains an optimal targetfor future therapies. In light of our findings, attempts to understandacquired resistance to other malignancies treated with STI-571, such asmetastatic gastrointestinal stromal tumors, might logically begin withsensitive sequence analysis of the c-Kit kinase domain. We envision thefuture of clinical management for CML to involve, in addition to theroutine usage of sensitive kinase domain mutation detection methods,combination molecular therapy, using multiple agents with the ability totarget Bcr-Abl as well as kinase-active STI-571-resistant isoforms inaddition to downstream effectors.

Typical embodiments of the invention are described below.

MARS Polynucleotides

A number of specific sequences of MARS are identified in Table I below.One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a MARS gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a MARS protein and fragments thereof, DNA, RNA, DNA/RNA hybrid,and related molecules, polynucleotides or oligonucleotides complementaryto a MARS gene or mRNA sequence or a part thereof, and polynucleotidesor oligonucleotides that hybridize to a MARS gene, mRNA, or to a MARSencoding polynucleotide (collectively, “MARS polynucleotides”). As usedherein, the MARS gene and protein is meant to include the MARS genes andproteins specifically described herein and the genes and proteinscorresponding to MARS proteins. Typical embodiments of the inventiondisclosed herein include MARS polynucleotides containing specificportions of the MARS mRNA sequence (and those which are complementary tosuch sequences), for example, those that encode the T315I codonsequence.

Therefore, one specific aspect of the invention provides polynucleotidescorresponding or complementary to all or part of a T315I Bcr-Abl gene,mRNA, and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding a T315I Bcr-Abl protein and fragments thereof,DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a T315I Bcr-Abl gene or mRNA sequenceor a part thereof, and polynucleotides or oligonucleotides thathybridize to a T315I Bcr-Abl gene, mRNA, or to a T315I Bcr-Abl encodingpolynucleotide (collectively, “T315I Bcr-Abl polynucleotides”). As usedherein, the T315I Bcr-Abl gene and protein is meant to include the T315IBcr-Abl genes and proteins specifically described herein and the genesand proteins corresponding to T315I Bcr-Abl proteins. Typicalembodiments of the invention disclosed herein include T315I Bcr-Ablpolynucleotides containing specific portions of the T315I Bcr-Abl mRNAsequence (and those which are complementary to such sequences), forexample, those that encode the T315I codon.

The MARS polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their in the detection of theMARS gene(s), mRNA(s), or fragments thereof; as reagents for thediagnosis and/or prognosis of cancers; as coding sequences capable ofdirecting the expression of MARS polypeptides; as tools for modulatingor inhibiting the function of the MARS protein.

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of theMARS polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a MARS polynucleotide in a sample and as ameans for detecting a cell expressing a MARS protein.

Examples of such probes and primers include polypeptides comprising allor part of a human MARS cDNA sequence shown in Table I. Examples ofprimer pairs capable of specifically amplifying MARS mRNAs (e.g. thoseprimers disclosed herein) are readily ascertainable by those skilled inthe art. As will be understood by the skilled artisan, a great manydifferent primers and probes may be prepared based on the sequencesprovided in herein and used effectively to amplify and/or detect a MARSmRNA.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga MARS polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a MARS polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 cell). Examples of suitablemammalian cells include various cancer cell lines, other transfectableor transducible cell lines, including those mammalian cells routinelyused for the expression of recombinant proteins (e.g., COS, CHO, 293,293T cells etc.). More particularly, a polynucleotide comprising thecoding sequence of a MARS may be used to generate MARS proteins orfragments thereof using any number of host vector systems routinely usedand widely known in the art.

A wide range of host vector systems suitable for the expression of MARSproteins or fragments thereof are available, see for example, Sambrooket al., 1989, supra; Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expressionvectors, MARS may be preferably expressed in cell lines, including forexample CHO COS, 293, 293T, rat-1, 3T3 etc. The host vector systems ofthe invention are useful for the production of a MARS protein orfragment thereof. Such host-vector systems may be employed to study thefunctional properties of MARS and MARS mutations.

MARS Polypeptides

Another aspect of the present invention provides MARS proteins andpolypeptide fragments thereof. The MARS proteins of the inventioninclude those specifically identified herein. Fusion proteins thatcombine parts of different MARS proteins or fragments thereof, as wellas fusion proteins of a MARS protein and a heterologous polypeptide arealso included. Such MARS proteins will be collectively referred to asthe MARS proteins, the proteins of the invention, or MARS. As usedherein, the term “MARS polypeptide” refers to a polypeptide fragment ora MARS protein of at least about 6 amino acids (e.g. a Bcr-Ablpolypeptide having about 6 contiguous amino acids including a MARS suchas T315I, preferably at least about 10-15 amino acids).

Proteins encoded by the MARS genes, or by fragments thereof, will have avariety of uses, including but not limited to generating antibodies andin methods for identifying ligands and other agents (e.g. smallmolecules such as 2-phenylpyrimidines) and cellular constituents thatbind to a MARS gene product. Antibodies raised against a MARS protein orfragment thereof may be useful in diagnostic and prognostic assays,imaging methodologies (including, particularly, cancer imaging), andtherapeutic methods in the management of human cancers characterized byexpression of a MARS protein, including but not limited to cancer of thelymphoid lineages. Various immunological assays useful for the detectionof MARS proteins are contemplated, including but not limited to varioustypes of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting leukemia cells(e.g., in radioscintigraphic imaging methods).

MARS Antibodies

The term “antibody” is used in the broadest sense and specificallycovers single anti-MARS monoclonal antibodies (including agonist,antagonist and neutralizing antibodies) and anti-MARS antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” (mAb) as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiescomprising the individual population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

Another aspect of the invention provides antibodies thatimmunospecifically bind to MARS proteins and polypeptides. The mostpreferred antibodies will specifically bind to a MARS protein and willnot bind (or will bind weakly) to Bcr-Abl proteins and polypeptides.Anti-MARS antibodies that are particularly contemplated includemonoclonal and polyclonal antibodies as well as fragments containing theantigen binding domain and/or one or more complementarity determiningregions of these antibodies. As used herein, an antibody fragment isdefined as at least a portion of the variable region of theimmunoglobulin molecule that binds to its target, i.e., the antigenbinding region.

For some applications, it may be desirable to generate antibodies whichspecifically react with a particular MARS protein and/or an epitopewithin a particular structural domain. For example, preferred antibodiesuseful for diagnostic purposes are those which react with an epitope ina mutated region of the MARS protein as expressed in cancer cells. Suchantibodies may be generated by using the MARS proteins described herein,or using peptides derived from various domains thereof, as an immunogen.

MARS antibodies of the invention may be particularly useful in cancer(e.g. chronic myelogenous leukemia) therapeutic strategies, diagnosticand prognostic assays, and imaging methodologies. Similarly, suchantibodies may be useful in the diagnosis, and/or prognosis of othercancers, to the extent MARS is also expressed or overexpressed in othertypes of cancer. The invention provides various immunological assaysuseful for the detection and quantification of MARS and mutant MARSproteins and polypeptides. Such assays generally comprise one or moreMARS antibodies capable of recognizing and binding a MARS or mutant MARSprotein, as appropriate, and may be performed within variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting cancer cells are also provided by the invention,including but limited to radioscintigraphic imaging methods usinglabeled MARS antibodies. Such assays may be used clinically in thedetection, monitoring, and prognosis of cancers, particularly chronicmyeloid leukemia.

Mars Transgenic Animals

Nucleic acids that encode MARS can also be used to generate eithertransgenic animals which, in turn, are useful in the development andscreening of therapeutically useful reagents. A transgenic animal (e.g.,a mouse or rat) is an animal having cells that contain a transgene,which transgene was introduced into the animal or an ancestor of theanimal at a prenatal, e.g., an embryonic stage. A transgene is a DNAthat is integrated into the genome of a cell from which a transgenicanimal develops. In one embodiment, cDNA encoding T315I Bcr-Abl can beused to clone genomic DNA encoding T315I Bcr-Abl in accordance withestablished techniques and the genomic sequences used to generatetransgenic animals that contain cells that express DNA encoding T315IBcr-Abl. Methods for generating transgenic animals, particularly animalssuch as mice or rats, have become conventional in the art and aredescribed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for MARS transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding MARS introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding MARS. Such animals can beused as tester animals for reagents thought to confer protection from,for example, pathological conditions associated with its expression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Methods for the Detection of MARS

Another aspect of the present invention relates to methods for detectingMARS polynucleotides and MARS proteins, as well as methods foridentifying a cell that expresses MARS. The expression profile of MARSmakes them diagnostic markets for disease states. As discussed in detailbelow, the status of MARS gene products in patient samples may beanalyzed by a variety protocols that are well known in the art includingimmunohistochemical analysis, the variety of Northern blottingtechniques including in situ hybridization, RT-PCR analysis (for exampleon laser capture micro-dissected samples), western blot analysis andtissue array analysis.

More particularly, the invention provides assays for the detection ofMARS polynucleotides in a biological sample, such as cell preparations,and the like. A number of methods for amplifying and/or detecting thepresence of MARS polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a MARS mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a MARSpolynucleotides as sense and antisense primers to amplify MARS cDNAstherein; and detecting the presence of the amplified MARS cDNA. Anynumber of appropriate sense and antisense probe combinations may bedesigned from the nucleotide sequences provided for the MARS and usedfor this purpose.

The invention also provides assays for detecting the presence of a MARSprotein in a biological sample. Methods for detecting a MARS protein arealso well known and include, for example, immunoprecipitation,immunohistochemical analysis, Western Blot analysis, molecular bindingassays, ELISA, ELIFA and the like. For example, in one embodiment, amethod of detecting the presence of a MARS protein in a biologicalsample comprises first contacting the sample with a MARS antibody, aMARS-reactive fragment thereof, or a recombinant protein containing anantigen binding region of a MARS antibody; and then detecting thebinding of MARS protein in the sample thereto.

Methods for identifying a cell that expresses MARS are also provided. Inone embodiment, an assay for identifying a cell that expresses a MARSgene comprises detecting the presence of MARS mRNA in the cell. Methodsfor the detection of particular mRNAs in cells are well known andinclude, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled MARS riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for MARS, and other amplification type detection methods, suchas, for example, branched DNA, SISBA, TMA and the like).

A significant aspect of the invention disclosed herein is the discoverythat amino acid substitutions in the Bcr-Abl polypeptide sequence shownin SEQ ID NO: 1 can produce cancer cells having a resistance to tyrosinekinase inhibitors such as STI-571. Specifically those skilled in thatart understand that the physiological mechanisms of drug resistance arediverse and that drug resistance typically occurs through othermechanisms such as an increase in the expression of proteins that exportthe drug out of the cell (see, e.g. Suzuki et al., Curr Drug Metab 2001December; 2(4):367-77). Consequently, the disclosure herein provides thescientific evidence to confirm the Bcr-Abl polypeptide sequence shown inSEQ ID NO: 1 as a target for analysis in methods relating to identifyingdrug resistant cells, such as methods of identifying an amino acidsubstitution in at least one Bcr-Abl polypeptide expressed in humancancer cell from an individual selected for treatment with a tyrosinekinase inhibitor.

A preferred embodiment of the invention is a method of identifying atleast one amino acid substitution in at least one Bcr-Abl polypeptidehaving some level of tyrosine kinase activity that is expressed in ahuman cancer cell from an individual selected for treatment with atyrosine kinase inhibitor, the method comprising determining thepolypeptide sequence of at least one Bcr-Abl polypeptide expressed bythe human cancer cell and comparing the polypeptide sequence of theBcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1 so that an amino acidsubstitution in the Bcr-Abl polypeptide expressed by the human cancercell can be identified. In preferred methods of the invention, an aminoacid substitution so identified confers some level of resistance toSTI-571.

A significant aspect of the invention disclosed herein is thedelineation of a discreet region in the Bcr-Abl polypeptide sequenceshown in SEQ ID NO: 1 that contains mutations that can produce cancercells having a resistance to tyrosine kinase inhibitors such as STI-571.This discovery allows artisans to focus on this region in diagnosticprotocols so as to facilitate such analyses. In this context, apreferred method of the invention is a method of identifying an aminoacid substitution in at least one Bcr-Abl polypeptide expressed in ahuman cancer cell from an individual selected for treatment with atyrosine kinase inhibitor, the method comprising determining thepolypeptide sequence of at least one Bcr-Abl polypeptide expressed bythe human cancer cell and comparing the polypeptide sequence of theBcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1 so that an amino acidsubstitution in the Bcr-Abl polypeptide expressed by the human cancercell can be identified, wherein the amino acid substitution occurs in aregion of the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1comprising residue D233 through residue T406. Without being bound by aspecific scientific theory, the data disclosed herein provides evidencethat this region defines boundaries for the structural architecture ofthe portions of Bcr-Abl that are predominantly involved in aninteraction with STI-571.

Another significant aspect of the invention disclosed herein is thedelineation of a discreet subregions in the Bcr-Abl polypeptide sequenceshown in SEQ ID NO: 1 that contains the mutations that can producecancer cells having a resistance to tyrosine kinase inhibitors such asSTI-571. This discovery allows artisans to focus on such subregions indiagnostic protocols so as to facilitate such analyses. In this context,a preferred method of the invention is a method of identifying an aminoacid substitution in at least one Bcr-Abl polypeptide expressed in ahuman cancer cell from an individual selected for treatment with atyrosine kinase inhibitor, the method comprising determining thepolypeptide sequence of at least one Bcr-Abl polypeptide expressed bythe human cancer cell and comparing the polypeptide sequence of theBcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1 so that an amino acidsubstitution in the Bcr-Abl polypeptide expressed by the human cancercell can be identified, wherein the amino acid substitution occurs inthe P-loop (residue G249 through residue V256 of the Bcr-Abl polypeptidesequence shown in SEQ ID NO: 1), helix C (residue E279 through residueI293 of the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1), thecatalytic domain (residue H361 through residue R367 of the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1) or the activation loop(residue A380 through residue P402 of the Bcr-Abl polypeptide sequenceshown in SEQ ID NO: 1). Alternatively, the amino acid substitution isproximal (e.g. within about 10 amino acid residues) to one of thesesubregions in a manner that perturbs the function of the subregion.

A particularly significant aspect of the invention disclosed herein isthe delineation of a discreet residue positions in the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1 that, when mutated, canproduce cancer cells having a resistance to tyrosine kinase inhibitorssuch as STI-571. This discovery allows artisans to focus on such residuepositions in diagnostic protocols so as to facilitate such analyses. Inthis context, a preferred method of the invention is a method ofidentifying an amino acid substitution in at least one Bcr-Ablpolypeptide expressed in a human cancer cell from an individual selectedfor treatment with a tyrosine kinase inhibitor, the method comprisingdetermining the polypeptide sequence of at least one Bcr-Abl polypeptideexpressed by the human cancer cell and comparing the polypeptidesequence of the Bcr-Abl polypeptide expressed by the human cancer cellto the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1 so that anamino acid substitution in the Bcr-Abl polypeptide expressed by thehuman cancer cell can be identified, wherein the amino acid substitutionoccurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253,E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274,D276, T277, M278, E282, F283, A288, M290, K291, E292, I293, P296, L298,V299, Q300, G303, V304, C305, T306, F311, I314, T315, E316, F317, M318,Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342,M343, A344, I347, A350, M351, E352, E355, K357, N358, F359, I360, L364,E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396,A399, P402, or T406.

This identification of discreet residue positions in the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1 that, when mutated, canproduce cancer cells having a resistance to tyrosine kinase inhibitorssuch as STI-571 is significant in part because of art which teaches thatin situations where methodical experimentation has established that theproperties of a specific residue at a particular position within thepolypeptide chain are crucial for maintaining some aspect of a protein'sfunctional integrity, an alteration in the size, shape, charge,hydrogen-bonding capacity or chemical reactivity of the amino acid sidechain at one of these “active” amino acid positions is likely to affectthe properties of the protein in some way (See e.g. Rudiger et al.,Peptide Hormones, University Park Press (1976)). For this reason, theskilled artisan would reasonably expect a substitution in a residueshown to be important for the inhibition of tyrosine kinase activity bySTI-571 in the wild type protein to effect the ability of STI-571 toinhibit the kinase activity of the Bcr-Abl polypeptide. As disclosedherein, the specific effects of any substitution mutation (or atruncation, a deletion, a frame shift etc.) on STI-571 resistance can beexamined by protocols such as those disclosed in the examples below.

In specific embodiments of the methods disclosed herein, the amino acidsubstitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F,Y253H, E255K, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R,V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E82G, F283S,A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L,Q300R, G303E, V304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A,T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P,R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V,I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V,I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G,F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T or T406A. Whilethe identification of substitutions is a preferred embodiment of theinvention, the methods disclosed herein can also be used to identifyother mutations that are associated with resistance to tyrosine kinaseinhibitors such as STI-571 such as truncations that result from amutation that introduces a stop codon at an amino acid residue positionsuch as K245STOP or E334STOP.

Embodiments of the invention include those that examine any one to allof the amino acid positions in the Bcr-Abl polypeptide sequence (e.g.M1, L2, E3 through V1128, Q1129 and R1130) as occurs when one comparesthe sequence of a polypeptide expressed by a cancer cell with thepolypeptide sequence shown in SEQ ID NO: 1. In this context, inpreferred embodiments of the invention, one can examine residue G250,Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343,M343, A344, M351T, E35, K357, I360, V379 or H396. In specificembodiments one can examine residue G250, Q252, Y253, E255, T315, F317,M351 or E355.

As is known in the art, it may be desirable to examine one residue butnot necessarily all of the amino acid positions in the Bcr-Ablpolypeptide sequence. Consequently, another embodiment of the inventionis a method of identifying an amino acid substitution in at least oneBcr-Abl polypeptide expressed in a human cancer cell from an individualselected for treatment with a tyrosine kinase inhibitor, the methodcomprising determining the polypeptide sequence of at least one Bcr-Ablpolypeptide expressed by the human cancer cell and comparing thepolypeptide sequence of the Bcr-Abl polypeptide expressed by the humancancer cell to the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1 sothat an amino acid substitution in the Bcr-Abl polypeptide expressed bythe human cancer cell can be identified, wherein the amino acidsubstitution does not occur at residue G250, Q252, E255, K264, V270,F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35,K357, I360, V379 or H396. Corresponding embodiments of the inventioninclude those that examine one or more amino acid mutations in a Bcr-Ablpolypeptide but do not examine another specific amino acid position inthe Bcr-Abl polypeptide sequence (e.g. methods which examine residueposition 315 but not residue position 255).

The polynucleotide and/or polypeptide sequences of Bcr-Abl can beidentified by any one of a wide variety of protocols known in the artsuch as those disclosed herein. In preferred methods, the Bcr-Ablpolynucleotide expressed by the human cancer cell is isolated by thepolymerase chain reaction. In addition, methods used in theidentification of one Bcr-Abl polypeptide expressed in a human cancercell from an individual selected for treatment with one tyrosine kinaseinhibitor can be identical to methods used in the identification of oneBcr-Abl polypeptide expressed in a human cancer cell from an individualselected for treatment with another tyrosine kinase inhibitor. Inillustrative methods of the invention, the kinase inhibitor is a2-phenylaminopyrimidine.

As noted herein, the methods of the present invention can be used indetermining whether or not to treat an individual with a specifictyrosine kinase inhibitor such as STI-571. Another embodiment of theinvention disclosed herein is a method of identifying a mutation in aBcr-Abl polynucleotide in a mammalian cell, wherein the mutation in aBcr-Abl polynucleotide is associated with resistance to inhibition ofBcr-Abl tyrosine kinase activity by a 2-phenylaminopyrimidine, themethod comprising determining the sequence of at least one Bcr-Ablpolynucleotide expressed by the mammalian cell and comparing thesequence of the Bcr-Abl polynucleotide to the Bcr-Abl polynucleotidesequence encoding the polypeptide sequence shown in SEQ ID NO: 1,wherein the mutation in the Bcr-Abl polynucleotide comprises analteration at amino acid residue position: D233, T243, M244, K245, G249,G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265,V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291,E292, I293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, I314,T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333,E334, A337, V339, L342, M343, A344, I347, A350, M351, E352, E355, K357,N358, F359, I360, L364, E373, N374, K378, V379, A380, D381, F382, T389,T392, T394, A395, H396, A399, P402, or T406 of the polypeptide sequenceshown in SEQ ID NO: 1. As used herein, “a Bcr-Abl polynucleotideassociated with resistance to inhibition of Bcr-Abl tyrosine kinase by a2-phenylaminopyrimidine” refers to a Bcr-Abl polynucleotide that hasbeen identified in cancer cells that exhibit some level of resistance toa 2-phenylaminopyrimidine such as STI-571 (or analogs or derivativesthereof) and which encodes a polypeptide having at least one amino aciddifference from the polypeptide sequence shown in SEQ ID NO: 1 (e.g.those disclosed in Table IA). Preferably the Bcr-Abl polynucleotideassociated with resistance to inhibition of Bcr-Abl tyrosine kinase by a2-phenylaminopyrimidine encodes a polypeptide that exhibits exhibit somelevel of resistance to a 2-phenylaminopyrimidine such as STI-571.

Optionally, in the methods disclosed above, the mammalian cell is ahuman cancer cell. In preferred methods, the human cancer cell is achronic myeloid leukemia cell. In highly preferred methods, the humancancer cell is obtained from an individual treated with STI-571.Optionally, the amino acid substitution in the Bcr-Abl polypeptideexpressed in human cancer cell confers resistance to inhibition oftyrosine kinase activity by STI-571.

MARS expression analysis may also be useful as a tool for identifyingand evaluating agents that modulate MARS gene expression. Identificationof a molecule or biological agent that could inhibit MARS activity is oftherapeutic value.

Monitoring the Status of MARS

The finding that MARS mRNA is expressed in cancers demonstrating STI-571resistance provides evidence that mutations in Bcr-Abl are associatedwith STI-571 resistance and therefore identifies these genes and theirproducts as targets that the skilled artisan can use to evaluatebiological samples from individuals suspected of having a diseaseassociated with MARS expression. In this context, the evaluation of thestatus of MARS genes and their products can be used to gain informationon the disease potential of a tissue sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition a gene and its products including,but not limited to the integrity and/or methylation of a gene includingits regulatory sequences, the location of expressed gene products(including the location of MARS expressing cells), the presence, level(e.g. the percentage of MARS expressing myeloid cancer cells in a totalpopulation of myeloid cancer cells), and biological activity ofexpressed gene products (such as MARS mRNA polynucleotides andpolypeptides), the presence or absence of transcriptional andtranslational modifications to expressed gene products as well asassociations of expressed gene products with other biological moleculessuch as protein binding partners. The status of MARS can be evaluated bya wide variety of methodologies well known in the art, typically thosediscussed below.

The status of MARS may provide information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining MARS status and diagnosing cancers that express MARS. MARSstatus in patient samples may be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, western blot analysis of clinical samples andcell lines, and tissue array analysis. Typical protocols for evaluatingthe status of the MARS gene and gene products can be found, for examplein Ausubul et al. eds., 1995, Current Protocols In Molecular Biology,Units 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting]and 18 [PCR Analysis].

A typical aspect of the invention is directed to assessing theeffectiveness of STI-571 in a therapeutic regimen. In a representativeembodiment, a method for assessing the effectiveness of STI-571comprises detecting MARS mRNA or MARS protein in a tissue sample, itspresence indicating a likely resistance to STI-571, wherein the degreeof MARS mRNA expression (e.g. the percentage of clones that express oneor more MARS) is proportional to the likelihood of resistance toSTI-571.

Another aspect of the invention is directed to examining the stage ofcancer in an individual. In one embodiment, a method for examining astage of cancer comprises detecting MARS mRNA or MARS protein in atissue sample, its presence indicating susceptibility to cancer, whereinthe degree of MARS mRNA expression present is proportional to the degreeof susceptibility. In a specific embodiment, the presence of MARS in atissue sample is examined, with the presence of MARS in the sampleproviding an indication of a stage of leukemia (or the emergence orexistence of a leukemia). In a closely related embodiment, one canevaluate the integrity MARS nucleotide and amino acid sequences in abiological sample in order to identify perturbations in the structure ofthese molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in MARS geneproducts in the sample providing an indication of cancer stage orsusceptibility (or the emergence or existence of a cancer type orstage).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of MARS mRNAor MARS protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of MARS mRNA or MARS proteinexpressed in a corresponding control tissue, wherein the degree of MARSmRNA expression present is proportional to the degree of aggressiveness.In a specific embodiment, aggressiveness of leukemias is evaluated bydetermining the extent to which MARS is expressed in the tumor cells,with relatively higher numbers of cells expressing one or more MARSindicating more aggressive tumors (e.g. in that they are resistant to atherapeutic agent such as STI-571).

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of MARS mRNA orMARS protein expressed by cells in a sample of the tumor, comparing thelevel so determined to the level of MARS mRNA or MARS protein expressedin an equivalent tissue sample taken from the same individual at adifferent time, wherein the degree of MARS mRNA or MARS proteinexpression in the tumor sample over time provides information on theprogression of the cancer. In a specific embodiment, the progression ofa cancer is evaluated by determining the extent to which MARS expressionin the tumor cells alters over time, with higher expression levels overtime indicating a progression of the cancer.

Gene amplification provides an additional method of assessing the statusof Bcr-Abl. Gene amplification may be measured in a sample directly, forexample, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

The above diagnostic approaches may be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof MARS gene and/or MARS gene products and a factor that is associatedwith malignancy as a means of diagnosing and prognosticating the statusof a tissue sample. In this context, a wide variety of factorsassociated with malignancy may be utilized such as the expression ofgenes otherwise associated with malignancy as well as gross cytologicalobservations (see e.g. Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Eptsein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of MARS gene and MARS gene products and an additional factorthat is associated with malignancy are useful, for example, because thepresence of a set or constellation of specific factors that coincideprovides information crucial for diagnosing and prognosticating thestatus of a tissue sample.

In a typical embodiment, methods for observing a coincidence between theexpression of MARS gene and MARS gene products (or perturbations in MARSgene and MARS gene products) and a factor that is associated withmalignancy entails detecting the overexpression of MARS mRNA or proteinin a tissue sample and then detecting the altered expression of anotheroncogene such RAS, or a tumor suppressor such as p53 or Rb, in a tissuesample, and observing a coincidence of MARS mRNA or protein expressionand, for example, RAS mRNA or protein overexpression. In a specificembodiment, the expression of MARS and RAS mRNA in tissue is examined.In a preferred embodiment, the coincidence of MARS and RAS mRNAoverexpression in the sample provides an indication of leukemia stage,or the emergence or existence of a leukemia.

Preferred embodiments of the invention described herein include methodsfor characterizing a cancer genotype and/or phenotype such as thegenotype and/or phenotype of cancers of the myeloid lineage. Specificembodiments of the invention described herein include methods ofassessing the likelihood of resistance to a nucleotide analog such as2-phenylamino pyrimidine. Particular embodiments of the inventiondescribed herein include methods for specifically identifying cellshaving some degree of resistance to STI-571. Such methods typicallyinclude the step of sequencing a target kinase such as Bcr-Abl toidentify a mutation associated with a specific genotype or phenotypesuch as resistance to STI-571. Preferably the mutation is within adomain shown to be associated with the cancer genotype and/or phenotype(e.g. the ATP binding domain of Bcr-Abl). More preferably the mutationis in a Bcr-Abl residue identified in Table I below (or in an equivalentresidue of a kinase having homology to Bcr-Abl).

A variety of permutations of these methods are provided by the inventiondisclosed herein. For example, the invention disclosed herein allowsartisans to examine MARS in a variety of contexts to determine whetherdifferent mutations segregate with specific clinical phenotypes (e.g.lymphoid versus myeloid disease) or with different clinical patterns ofSTI-571 resistance (e.g. refractory disease; delayed relapse versusrapid relapse). The invention further allows those skilled in the art todetermine whether kinase domain mutations restricted to patients withadvanced stage disease or also occur in chronic phase patients. Theinvention also allows those skilled in the art to determine whether oneor more mutations are a manifestation of the clonal diversity andgenetic instability associated with disease progression. The inventionalso allows those skilled in the art to determine whether such mutationsare a consequence of prior exposure to chemotherapy, or occur only inpatients exposed to STI-571. The invention also allows those skilled inthe art to determine the biological implications for other targetedkinase inhibitors currently in clinical development.

Methods for detecting and quantifying the expression of MARS mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of MARS mRNAinclude in situ hybridization using labeled MARS riboprobes, northernblot and related techniques using MARS polynucleotide probes, RT-PCRanalysis using primers specific for MARS, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RT-PCR may be usedto detect and quantify MARS mRNA expression as described in the Examplesthat follow. Any number of primers capable of amplifying MARS may beused for this purpose, including but not limited to the various primersets specifically described herein. Standard methods for the detectionand quantification of protein may be used for this purpose. In aspecific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the MARS protein may be used in an immunohistochemicalassays of samples. Antibodies directed against MARS protein can also beused to detect MARS in a patient specimen (e.g., blood or other sample)using conventional techniques such as fluorescence-activated cellsorting (FACS) and/or ELISA.

As discussed in detail below, once a mutant sequence is identified onecan then identify compounds which bind and/or inhibit the activity ofthe mutant kinases.

Methods for Identifying and Characterizing MARS

The disclosure provided herein allows those skilled in the art toidentify and characterize cells having a genotype and/or phenotypeassociated with a cancer such as a genotype and/or phenotype associatedwith cancers of the myeloid lineage. Specific embodiments of theinvention described herein include methods for the identification andcharacterization of Bcr-Abl mutants associated resistance to anucleotide analog such as 2-phenylamino pyrimidine. Particularembodiments of the invention described herein include methods for theidentification and characterization of cells having some degree ofresistance to an inhibitor such as STI-571.

A first method for characterizing cells having a genotype and/orphenotype associated with a cancer includes the sequencing of Bcr-Abl inthose cells to identify one or more mutations associated with aparticular phenotype (e.g. resistance to STI-571) such as a mutation ina domain or region shown to be associated with a specific genotypeand/or phenotype (e.g. the ATP binding domain of Bcr-Abl). Preferablythe mutation is in a Bcr-Abl residue identified in Table I below.

A related method for characterizing cells having a genotype and/orphenotype associated with a cancer and/or cancer stage includesconsidering the location of the mutation in the context of the crystalstructure of the ABL kinase domain bound to a variant STI-571 (see, e.g.Schindler et al., Science. 2000 Sep. 15; 289(5486):1938-42). Thisdefinition of the crystal structure allows one to evaluate whether themutation might interfere with the anti-leukemia activity of STI-571.Based on this analysis, one can prioritize mutations for directexperimental analysis of ABL kinase activity, leukemogenicity and levelof inhibition by STI-571.

Another method for characterizing cells having a genotype and/orphenotype associated with a cancer and/or cancer stage includesanalyzing another factor associated a genotype and/or phenotypeassociated with a cancer in a target cell being examined such as thestage of the disease progression, the relative frequency of the mutantwithin the population (e.g. is the clone a dominant population whichprovides evidence that they have a growth advantage).

Another method for characterizing cells having a genotype and/orphenotype associated with a cancer includes engineering selectedmutations into wild-type BCR-ABL cDNA to create a mutant allele whoseenzymological and biological properties can be examined directly.Enzymology can be performed by measuring tyrosine kinase activity invitro or in cells using standard assays known in the art (see, e.g.those cited in Example 1). Biological activity can be measured usingstandard oncogene transformation assays using growth factor dependenthematopoietic cell lines or primary mouse bone marrow cells (see, e.g.those cited in Example 1). In this way, resistance to STI-571 can bemeasured using such kinase assays and transformation assays.

Those skilled in the art will understand that the above described assaysfor characterizing cells having a genotype and/or phenotype associatedwith a cancer can be performed independently or in combination with eachother.

Mutations in Related Molecules

Residues shown to mutated in MARS occur in domains that are highlyconserved among members of the protein kinase family (see, e.g. Hanks etal., Science 241: 42-51 (1988)). The finding that a highly conservedresidue is mutated in cancers and that this mutation is associated withresistance to a chemotherapeutic agent provides evidence that thisdomain is associated with dysregulated cell growth and thereforeidentifies these domains and residue position as a targets that theskilled artisan can use to evaluate the status of related members of thetyrosine kinase family (see, e.g. those identified in FIG. 1 of Hanks etal., Science 241: 42-51 (1988)), from individuals suspected of having adisease associated with the dysregulation of that member of the tyrosinekinase family.

In this context, the evaluation of the status of a domain and/or residuein the tyrosine kinase family member can be used to gain information onthe disease potential of a tissue sample. For example, in a syndrome inwhich the dysregulation of a specific tyrosine kinase family member isknown or suspected (preferably one that exhibits a pattern of pathologythat is similar to that seen with Bcr-Abl), one can determine if amutation has occurred at that residue in order to obtain evidence ofgenetic changes associated with growth dysregulation (e.g. resistance toa chemotherapeutic agent). Methods for the detection of mRNAs havingsuch specific mutations in cells are well known and include, forexample, hybridization assays using complementary DNA probes (such as insitu hybridization, Northern blot and related techniques) and variousnucleic acid amplification assays (such as RT-PCR using complementaryprimers specific for the mRNA of interest, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). As discussed below, methods for identifying molecules thatinteract with such mutant members of the tyrosine kinase family are alsoprovided.

Embodiments of the invention include methods for identifying afunctional hotspot (e.g. a region in a protein which has significantfunctional importance in kinase activity and drug resistance) in atarget kinase comprising sequencing at least a portion of the targetkinase to identify a mutation and comparing the location of the mutationto the location of functional hotspots identified in a homologous kinase(e.g. Bcr-Abl), wherein the identification of a mutation in a targetkinase that corresponds to a hotspot in a homologous kinase providesevidence that the mutation in the target kinase is in a functionalhotspot. Typically the hotspot occurs in a Bcr-Abl domain havingmutations associated with STI-571 resistance (e.g. the activation loop).More preferably the hotspot occurs in a Bcr-Abl residue identified inTable I. Preferably, the homologous kinase is Bcr-Abl and the homologiesare compared via a BLAST analysis. The target kinase may be any one of awide variety of kinases known in the art such as c-kit, PDGFR, EGFR andVEGFR or one of the kinases identified in FIG. 1 of Hanks et al.,Science 241: 42-51 (1988). Optionally these methods can be used tocharacterize cells from patients suffering from a pathology associatedwith aberrant expression of the target kinase.

Other embodiments of the invention include methods for assessing thelikelihood of a target kinase having a resistance to a nucleotide analogsuch as 2-phenylamino pyrimidine comprising sequencing at least aportion of the target kinase to identify a mutation, wherein theidentification of a mutation in a target kinase that corresponds to ahotspot in a homologous kinase provides evidence that target kinase willbe resistant to the inhibitor. Preferably the hotspot occurs in aBcr-Abl domain having mutations associated with STI-571 resistance. Morepreferably the hotspot occurs in a Bcr-Abl residue identified in TableI. Preferably, the homologous kinase is Bcr-Abl and the homologies arecompared via a BLAST analysis. The target kinase may be any one of awide variety of kinases known in the art such as c-kit, PDGFR, EGFR andVEGFR or one of the kinases identified in FIG. 1 of Hanks et al.,Science 241: 42-51 (1988) which is incorporated herein by reference.Optionally these methods can be used to characterize cells from patientssuffering from a pathology associated with aberrant expression of thetarget kinase.

The invention disclosed herein includes the identification of amino acidresidues in Bcr-Abl that are mutated in a manner characterized such thatthey retain kinase activity yet are associated with resistance toinhibition of kinase activity by a 2-phenylaminopyrimidine. Oneembodiment of an invention provided by this disclosure is a method ofidentifying such a mutation in an Abelson protein kinase, wherein themutation is associated with the resistance to an inhibition of kinaseactivity by a 2-phenylaminopyrimidine, the method comprising:determining an amino acid sequence of a portion of a polynucleotideencoding the Abelson protein kinase to determine the presence of amutation, wherein the mutation occurs at a amino acid residue at thesame relative position as a mutation in the C-Abl protein kinase shownin SEQ ID NO: 1 that is associated with STI-571 resistance as determinedusing the homology criteria of BLAST analysis. In this context, skilledartisans understand that mutations in the C-Abl protein kinase shown inSEQ ID NO: 1 that are associated with STI-571 resistance includemutations in the C-Abl protein kinase which have, for example, beenidentified in cancer cells isolated from individuals shown to exhibit aresistance to a therapeutic regime involving a 2-phenylaminopyrimidinesuch as STI-571. As disclosed herein, mutants of the C-Abl proteinkinase shown in SEQ ID NO: 1 that are identified as being associatedwith STI-571 resistance are readily characterized by any one of a widevariety of techniques that are well known in the art in view of theextensive biological characterization of c-Abl, Bcr-Abl and/or one ofthe Abelson protein kinases such as ARG etc. Such protocols includeanalyses based on the understanding of the biological significance of adomain or residue within these proteins that has been characterized ashaving significance in kinase activity or small molecule interaction(see, e.g. Example 3 below which identifies various previouslyidentified domains as well as residues which directly interact withSTI-571 via previously described crystallographic analyses etc). Suchprotocols further include biological analyses of biological activity ofthese mutants including for example, the well known assays forcharacterizing the kinase activities and transforming abilities ofAbelson protein kinases that are cited in Example 1 below.

A related embodiment is a method of identifying a mutant Abelson proteintyrosine kinase expressed by a cell by determining a nucleotide sequenceof a portion of a polynucleotide encoding the kinase domain of theAbelson protein tyrosine kinase expressed by the cell and then comparingthe nucleotide sequence so determined to that of the wild type sequenceof the Abelson protein tyrosine kinase to identify the presence of amutation, wherein the mutation so identified has the characteristics ofoccurring at a amino acid residue located within the polypeptidesequence of the Abelson protein tyrosine kinase at the same relativeposition as a mutation in the C-Abl protein kinase shown in SEQ ID NO: 1that has been identified as being associated with a resistance to aninhibition of tyrosine kinase activity by a 2-phenylaminopyrimidine, asdetermined using the homology parameters of a BLAST analysis (e.g. c-srcposition 338 which corresponds to position 315 in SEQ ID NO: 1). In aspecific version of this embodiment, the cell expressing the mutantAbelson protein tyrosine kinase is found in a population of cancer cellsthat has been observed in clinical populations to exhibit a resistanceto an inhibition of tyrosine kinase activity by a2-phenylaminopyrimidine (e.g. STI-571). In a highly preferred embodimentthe mutation in the C-Abl protein kinase shown in SEQ ID NO: 1 that hasbeen identified as being associated with a resistance to an inhibitionof tyrosine kinase activity by a 2-phenylaminopyrimidine is a Bcr-Ablresidue identified in Table I.

Yet another embodiment of the invention is a method of identifying amutant Abelson tyrosine kinase expressed by a cell by determining anucleotide sequence of a portion of the catalytic domain of the Abelsontyrosine kinase expressed by the cell (and more preferably thenucleotide binding site within the catalytic domain) and then comparingthe nucleotide sequence so determined to that of the wild type sequenceof the catalytic domain of the Abelson protein tyrosine kinase toidentify the presence of a mutation within the catalytic domain, whereinthe mutation so identified has the characteristics of occurring at aamino acid residue located within the polypeptide sequence of theAbelson protein tyrosine kinase at a amino acid residue that hashomology to an amino acid position in a C-Abl kinase shown in SEQ ID NO:1 that is associated with a resistance to an inhibition of tyrosinekinase activity by a 2-phenylaminopyrimidine, wherein the homologybetween the amino acid residue located within the polypeptide sequenceof the Abelson protein tyrosine kinase and the amino acid residue in theC-Abl kinase shown in SEQ ID NO: 1 that is associated with a resistanceto an inhibition of tyrosine kinase activity by a2-phenylaminopyrimidine can be illustrated via a BLAST analysis.

As used herein, an Abelson tyrosine kinase refers to the family ofkinases known in the art to be closely related to the c-Abl protein orhave domains that share a high degree of homology with a domain in thec-Abl protein. For example, the Philadelphia translocation is known toresult in the expression of a family of chimeric proteins in which aportion of the Bcr protein is fused to c-Abl protein. A specificgrouping of Abelson tyrosine kinase family members are those whichexhibit an amino acid sequence homology that is structurally and/orfunctionally related such that a 2-phenylaminopyrimidine can interactwith these molecules and inhibit their kinase activities (e.g. Bcr-Abl,TEL-Abl, c-kit, PDGFR, EGFR and VEGFR).

Another representative member of the Abelson tyrosine kinase family isthe protein designated ARG. An analysis of the amino acid sequence ofthe ARG protein reveals that it is closely related to that of c-Abl(see, e.g., Kruh et al., PNAS 1990, 87(15): 5802-6 and Wang et al.,Oncogene 1996, 13(7): 1379-85). Specifically, c-Abl and ARG arestrikingly similar with regard to overall structural architecture aswell as the amino acid sequences of their tyrosine kinase domains.Additional members of the family include for example, Dash, Nabl, andFes/Fps (see e.g. Hunter et al., Science 241, 42-51 (1988)).

As is known in the art, the Abelson tyrosine kinase family of proteinkinases contain a catalytic domain that has a highly conservedstructural and functional architecture (see, e.g. Sicheri et al., CurrOpin Struct Biol. 1997 December; 7(6):777-85; and Sicheri et al.,Nature. 1997 Feb. 13; 385(6617):602-9). Understandably, because regionswithin the catalytic domain of these tyrosine kinases are known to behighly conserved among members of this gene family, it is observed thatSTI-571 also interacts with representative members of this family suchas c-kit and PDGFR (see, e.g., Tuveson et al., Oncogene. 2001 Aug. 16;20(36):5054-8; Buchdunger et al., J Pharmacol Exp Ther. 2000 October;295(1):139-45; Wang et al., Oncogene. 2000 Jul. 20; 19(31):3521-8;Heinrich et al., Blood. 2000 Aug. 1; 96(3):925-32; and Carroll et al.,Blood. 1997 Dec. 15; 90(12):4947-52).

As noted above, the catalytic domains of these protein kinases have ahighly conserved structural and functional architecture which allows forthe interaction of compounds of the 2-phenylaminopyrimidine class ofmolecules to interact with this domain and further provides the basisfor a variety of comparative analyses as well as rational drug design(see, e.g., Traxler et al., Med Res Rev. 2001 November; 21(6):499-512;Traxler et al., J Med Chem. 1999 Mar. 25; 42(6):1018-26; and Parang etal., Nat Struct Biol. 2001 January; 8(1):37-41 Singh et al., J Med Chem.1997 Mar. 28; 40(7):1130-5 and Furet et al., J Comput Aided Mol Des.1995 December; 9(6):465-72. Moreover, because the crystal structure ofthe catalytic domain of Abl complexed 2-phenylaminopyrimidines such asvariants of STI-571 has been determined, this provides information as tohow this class of molecules interacts with these highly conservedregions within these kinases (see, e.g., Schindler et al., Science. 2000Sep. 15; 289(5486):1938-42). Such analyses are enhanced by the fact thatthe crystal structures of a number of other tyrosine kinase inhibitorshave also been determined (see, e.g., Schindler et al., Mol Cell. 1999May; 3(5):639-48; Mohammadi et al., EMBO J. 1998 Oct. 15;17(20):5896-904).

As disclosed herein, the domain comprising the ATP binding site isidentified as a region that is mutated in Bcr-Abl proteins exhibitingresistance to STI-571. Interestingly, other chemical classes of TKinhibitors are known to bind the ATP binding site including quinazolinesand pyrazolo-pyrrolo-pyridopyrimidines (see, e.g., Tian et al.,Biochemistry. 2001 Jun. 19; 40(24):7084-91; Fry et al., Science. 1994Aug. 19; 265(5175):1093-5; Rewcastle et al., J Med Chem. 1996 Feb. 16;39(4):918-28; Rewcastle et al., J Med Chem. 1995 Sep. 1; 38(18):3482-7;Toledo et al., Curr Med Chem. 1999 September; 6(9):775-805; and Bridgeset al., Curr Med Chem. 1999 September; 6(9):825-43). Consequently, TKinhibitors which bind an ATP binding site having a high homology to theATP binding site of Bcr-Abl (and mutants exhibiting resistance to suchinhibitors) can be analogously identified and characterized using thedisclosure provided herein.

The invention provided herein identifies specific regions withinconserved protein kinase family members that impart resistance to aclass of tyrosine kinase inhibitors, thereby identifying these regionsas the targets of the diagnostic protocols described herein. Inparticular, while certain amino acid residues known to be involved in aninteraction with kinase inhibitors such as 2-phenylaminopyrimidines havebeen identified, it was not known whether a mutation could occur at aresidue within a domain having a specific biological activity that wouldinhibit the interaction between the kinase and the kinase inhibitor yetallow the kinase to retain a biological activity associated with apathological condition, particularly in cases where the mutation isobserved in clinical specimens. The disclosure provided hereinidentifies specific target domains (e.g. the ATP-binding domain) withinprotein kinases in which amino acid mutations can occur that render thekinase resistant to kinase inhibitors such as 2-phenylaminopyrimidinesyet allow the kinase to retain a biological activity that is associatedwith a pathological condition (e.g. chronic myeloid leukemia). Byidentifying a specific region in protein kinases in which mutationshaving these dual characteristics occur, the disclosure provided hereinallows the skilled artisan to employ diagnostic procedures that aretailored to specifically analyze polynucleotides encoding these regions(e.g. in PCR protocols used to identify protein kinases likely to beresistant to kinase inhibitors). In this way, the disclosure providedherein can reduce the amount of experimentation necessary tocharacterize a mutant protein kinase that is associated with apathological condition. Such analyses are facilitated by the fact thatthese target domains are so highly conserved among a variety of proteinkinases they are readily identified, and therefore easily targeted inprotocols used to identify the presence of such mutations in thesedomains.

Typical embodiments of the invention include a method of identifying amutation in the catalytic domain of a target protein kinase comprisingdetermining the amino acid sequence of the catalytic domain andcomparing it to the wild type sequence of the target protein kinasecatalytic domain to identify a mutation therein, wherein the catalyticdomain of the target protein kinase has at least about 60, 70, 80, 85,90 or 95% homology to the catalytic domain of c-able catalytic domainshown in SEQ ID NO: 1. A related embodiment is a method of identifying amutation in the activation loop domain of a target protein kinasecomprising determining the amino acid sequence of the activation loopdomain and comparing it to the wild type sequence of the target proteinkinase activation loop domain to identify a mutation therein, whereinthe activation loop domain of the target protein kinase has at leastabout 60, 70, 80, 85, 90 or 95% homology to the activation loop domainof c-able activation loop domain shown in SEQ ID NO: 1. A relatedembodiment is a method of identifying a mutation in the nucleotidebinding pocket of a target protein kinase comprising determining theamino acid sequence of the nucleotide binding pocket and comparing it tothe wild type sequence of the target protein kinase nucleotide bindingpocket domain to identify a mutation therein, wherein the nucleotidebinding pocket domain of the target protein kinase has at least about60, 70, 80, 85, 90 or 95% homology to the nucleotide binding pocketdomain of c-able catalytic domain shown in SEQ ID NO: 1. A relatedembodiment is a method of identifying a mutation in a target tyrosinekinase that is likely to be associated with resistance to a tyrosinekinase inhibitor comprising determining the amino acid sequence of theP-loop, helix c, activation loop or catalytic sequences as well assequences within about 10 amino acids of the respective domain(s), andcomparing it to the wild type sequence of the target protein kinaseP-loop, helix c, activation loop or catalytic sequences as well assequences within about 10 amino acids of the respective domain(s) toidentify a mutation therein, wherein the P-loop, helix c, activationloop or catalytic sequences as well as sequences within about 10 aminoacids of the respective domain(s) of the target protein kinase has atleast about 60, 70, 80, 85, 90 or 95% homology to the P-loop, helix c,activation loop or catalytic sequences as well as sequences within about10 amino acids of these domains in c-Abl.

Another related embodiment is a method of isolating a polynucleotideencoding a mutated catalytic domain of a target protein kinasecomprising employing PCR to amplify the catalytic domain of a targetprotein kinase, wherein the target protein kinase exhibits a biologicalactivity that is associated with a pathological condition and whereinthe target protein kinase exhibits a resistance to tyrosine kinaseinhibitors, and wherein the catalytic domain of the target proteinkinase has at least about 60, 70, 80, 85, 90 or 95% homology to thecatalytic domain of c-able catalytic domain shown in SEQ ID NO: 1,comparing the polynucleotide sequence encoding the amino acid sequenceof the catalytic domain and comparing it to the polynucleotide sequenceencoding the amino acid sequence wild type amino acid sequence of thetarget protein kinase catalytic domain so that a polynucleotide encodinga mutated catalytic domain is identified.

In a specific embodiment of these methods, at least one amino acidresidue that is mutated in the domain has homology to a residueidentified in Table I. In another specific embodiment, the targetprotein kinase having the mutation exhibits a kinase activity that isassociated with a pathological condition (e.g. cancer). In anotherspecific embodiment, the kinase activity of the target protein kinasethat is associated with a pathological condition (e.g. cancer) isresistant to inhibition by a tyrosine kinase inhibitor. In anotherspecific embodiment, the kinase activity of the target protein kinasethat is associated with a pathological condition (e.g. cancer) isresistant to inhibition by a 2-phenylaminopyrimidine. In anotherspecific embodiment, the target protein kinase is shown in Table 2 ofHanks et al., Science 241: 42-51 (1988). In another specific embodiment,the target protein kinase is a Bcr-Abl, a TEL-Abl, a c-kit, a PDGFR, anEGFR, an VEGFR.

A related embodiment comprises a method of characterizing a property ofa protein tyrosine kinase, wherein the protein kinase has at least about60, 70, 80, 85, 90 or 95% homology to c-able shown in SEQ ID NO: 1comprising determining whether the protein tyrosine kinase exhibits anactivity that is associated with a pathological condition (e.g. via aprocedure identified herein or citations in the art), determiningwhether the protein tyrosine kinase exhibits resistance to a tyrosinekinase inhibitor (e.g. via a procedure identified herein or citations inthe art), determining an amino acid sequence of the protein tyrosinekinase, determining whether the amino acid sequence of the proteintyrosine kinase contains a mutated residue, determining whether themutated residue occurs in the catalytic domain, the activation loopand/or the ATP binding domain and/or determining whether the mutatedresidue has homology to a residue shown in Table I, wherein the presenceof a mutated residue occurring in the catalytic domain, the activationloop and/or the ATP binding domain and/or wherein the mutated residuehas homology to a residue shown in Table I provides evidence that themutation so identified inhibits the interaction between the kinase andthe kinase inhibitor yet allow the kinase to retain its kinase activity.In a specific embodiment, the kinase activity of the protein kinase thatis associated with a pathological condition is resistant to inhibitionby a 2-phenylaminopyrimidine. In another specific embodiment, theprotein kinase is a protein kinase shown in Table 2 of Hanks et al.,Science 241: 42-51 (1988). In another specific embodiment, the proteinkinase is a Bcr-Abl, a TEL-Abl, a c-kit, a PDGFR, an EGFR, an VEGFR.

Yet another embodiment of the invention is a method of identifying amutant Abelson protein tyrosine kinase expressed by a mammalian cancercell by determining a nucleotide sequence of a portion of apolynucleotide encoding the kinase domain of the Abelson proteintyrosine kinase expressed by the cell and then comparing the nucleotidesequence so determined to that of the wild type sequence of the Abelsonprotein tyrosine kinase to identify the presence of a amino acidsubstitution in the mutant Abelson protein tyrosine kinase, wherein anyamino acid substitution so identified has the characteristics ofoccurring at a amino acid residue located within the polypeptidesequence of the Abelson protein tyrosine kinase at the same relativeposition as an amino acid substitution in the C-Abl protein kinase shownin SEQ ID NO: 1 that has been identified as being associated with aresistance to an inhibition of tyrosine kinase activity by a2-phenylaminopyrimidine, as can be determined using the homologyparameters of a WU-BLAST-2 analysis. In preferred embodiments of theinvention, the mutant Abelson tyrosine kinase expressed by the cell is amutant c-Abl (see, e.g. NCBI Accession P00519), Bcr-Abl (see, e.g. NCBIAccession NP_(—)067585), PDGFR (see, e.g. NCBI Accession NP002600),c-kit (see, e.g. NCBI Accession CAA29458), TEL-Abl (see, e.g. NCBIAccession CAA84815), or TEL-PDGFR (see, e.g. NCBI Accession AAA19786). Arelated embodiment of the invention comprises repeating steps (a)-(b)another mammalian cancer cell obtained from a different individual; andthen cataloging the mutations found in the mutant Abelson proteintyrosine kinases present in the mammalian cancer cells. Preferably insuch methods, the cell expressing the mutant Abelson protein tyrosinekinase is found in a population of mammalian cancer cells that areobserved to exhibit a resistance to an inhibition of tyrosine kinaseactivity after exposure to a 2-phenylaminopyrimidine. In such methods,the mammalian cancer cell is can be a human cancer cell obtained from anindividual selected for treatment with a tyrosine kinase inhibitorcomprising a 2-phenylaminopyrimidine. Preferably, the amino acidsubstitution confers resistance to inhibition of tyrosine kinaseactivity by a 2-phenylaminopyrimidine.

In a specific embodiment of such methods, the mutation in the C-Ablprotein kinase shown in SEQ ID NO: 1 that has been identified as beingassociated with a resistance to an inhibition of tyrosine kinaseactivity by a 2-phenylaminopyrimidine occurs at the same relativeposition as amino acid residue D233, T243, M244, K245, G249, G250, G251,Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270,T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, I293,P296, L298, V299, Q300, G303, V304, C305, T306, F311, I314, T315, E316,F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337,V339, L342, M343, A344, I347, A350, M351, E352, E355, K357, N358, F359,I360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394,A395, H396, A399, P402, or T406. Typically, the amino acid substitutionoccurs at the same relative position as amino acid residue G250, Q252,E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343,A344, M351T, E35, K357, I360, V379 or H396.

The disclosure provided herein allows a mutant identified by one themethods disclosed herein to be further characterized. Specifically, byutilizing enzymological and/or biological assays described herein aswell as those known in the art (illustrated by those disclosed, forexample, in the Examples below), a mutant that is found to occur in aconserved target domain of a protein kinase can be readily characterizedto assess the biological significance of this mutation (e.g. renderingthe protein kinase resistant to kinase inhibitors such as2-phenylaminopyrimidines yet allowing the kinase to retain a biologicalactivity that is associated with a pathological condition). Moreover, inthe context of proteins in which a target protein is identified, thedisclosure herein of assays for the measurement of the phosphotyrosinecontent in an analogous fashion to the assays of Crkl, an adaptorprotein which is specifically and constitutively phosphorylated byBcr-Abl in CML cells (see, e.g., FIGS. 1 and 2).

In addition to the mutations identified in Table I, scanning amino acidanalysis can also be employed in comparative analyses of compounds suchas 2-phenylaminopyrimidines to identify the significance of one or moreamino acids which are structurally and/or functionally involved in theinteraction between Abelson tyrosine kinases and compounds such as2-phenylaminopyrimidines (see, e.g. U.S. Pat. Nos. 6,004,931 and5,506,107). Among the preferred scanning amino acids are relativelysmall, neutral amino acids. Such amino acids include alanine, glycine,serine, and cysteine. Alanine is typically a preferred scanning aminoacid among this group because it eliminates the side-chain beyond thebeta-carbon and is less likely to alter the main-chain conformation ofthe variant. Alanine is also typically preferred because it is the mostcommon amino acid. Further, it is frequently found in both buried andexposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does notyield adequate amounts of variant, an isosteric amino acid can be used.

Identification of Molecules that Interact with MARS

As illustrated in Example 8, the MARS protein and nucleic acid sequencesdisclosed herein allow a skilled artisan to identify proteins, smallmolecules and other agents that interact with MARS, as well as pathwaysactivated by MARS via any one of a variety of art accepted protocols.For example, using the disclosure provided herein, one can employmethods used in the art to evaluate the interaction between STI-571 andBcr-Abl to evaluate interactions between test molecules and MARS.

A representative embodiment of this invention comprises a method ofscreening for a molecule that interacts with an MARS amino acid sequencecomprising the steps of contacting a population of molecules with theMARS amino acid sequence, allowing the population of molecules and theMARS amino acid sequence to interact under conditions that facilitate aninteraction, determining the presence of a molecule that interacts withthe MARS amino acid sequence, and then separating molecules that do notinteract with the MARS amino acid sequence from molecules that do. In aspecific embodiment, the method further comprises purifying a moleculethat interacts with the MARS amino acid sequence. The identifiedmolecule can be used to modulate a function performed by MARS.

This embodiment of the invention is well suited to screen chemicallibraries for molecules which modulate, e.g., inhibit, antagonize, oragonize or mimic, the activity of BCR-ABL as measured by one of theassays disclosed herein. The chemical libraries can be peptidelibraries, peptidomimetic libraries, chemically synthesized libraries,recombinant, e.g., phage display libraries, and in vitrotranslation-based libraries, other non-peptide synthetic organiclibraries (e.g. libraries of 2-phenylaminopyrimidines, quinazolines orpyrazolo-pyrrolo-pyridopyrimidines and the like etc.).

Exemplary libraries are commercially available from several sources(ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In some cases,these chemical libraries are generated using combinatorial strategiesthat encode the identity of each member of the library on a substrate towhich the member compound is attached, thus allowing direct andimmediate identification of a molecule that is an effective modulator.Thus, in many combinatorial approaches, the position on a plate of acompound specifies that compound's composition. Also, in one example, asingle plate position may have from 1-20 chemicals that can be screenedby administration to a well containing the interactions of interest.Thus, if modulation is detected, smaller and smaller pools ofinteracting pairs can be assayed for the modulation activity. By suchmethods, many candidate molecules can be screened.

Many diversity libraries suitable for use are known in the art and canbe used to provide compounds to be tested according to the presentinvention. Alternatively, libraries can be constructed using standardmethods. Chemical (synthetic) libraries, recombinant expressionlibraries, or polysome-based libraries are exemplary types of librariesthat can be used.

In one embodiment, one can screen peptide libraries to identifymolecules that interact with MARS protein sequences. In such methods,peptides that bind to a molecule such as MARS are identified byscreening libraries that encode a random or controlled collection ofamino acids. Peptides encoded by the libraries are expressed as fusionproteins of bacteriophage coat proteins, the bacteriophage particles arethen screened against the protein of interest.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with MARS proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Small molecules and ligands that interact with MARS can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with a MARS's ability to mediatephosphorylation and de-phosphorylation.

A typical embodiment is a method of identifying a compound whichspecifically binds a MARS shown in Table I, wherein said MARS exhibitstyrosine kinase activity, comprising the steps of contacting said MARSwith a test compound under conditions favorable to binding; and thendetermining whether said test compound binds to said MARS so that acompound which binds to said MARS can be identified. As the interactionbetween various Abelson tyrosine kinases and a variety of test compoundshave been previously described, skilled artisans are familiar with theconditions conducive to binding. A specific embodiment of this aspect ofthe invention includes the steps of transfecting cells with a constructencoding the MARS, contacting said cells with said test compound that istagged or labelled with a detectable marker and then analyzing saidcells for the presence bound test compound. In contexts where thetransfected cells are observed to preferentially bind the test compoundas compared to cells that have not been transfected with a MARSconstruct, this indicates that the test compounds is binding to the MARSprotein expressed by those cells.

A test compound which binds said MARS may then be further screened forthe inhibition of a biological activity (e.g. tyrosine kinase activity)of said MARS. Such an embodiment includes, for example determiningwhether said test compound inhibits the tyrosine kinase activity of theMARS by utilizing molecular biological protocols to create recombinantcontracts whose enzymological and biological properties can be examineddirectly. A specific biological activity such as resistance to STI-571can be measured using standard kinase assays and transformation assays.Enzymology is performed for example, by measuring tyrosine kinaseactivity in vitro or in MARS expressing cells using standard assays(see, e.g. one of those cited in the Examples below). Alternatively,biological activity is measured using standard oncogene transformationassays (see, e.g. one of those cited in the Examples below).

A specific embodiment of the invention entails determining whether atest compound inhibits the biological activity of a MARS tyrosine kinaseinhibitor in a procedure that is analogous for examining how STI-571inhibits the tyrosine kinase activity of Bcr-Abl. Such methods typicallycomprise the steps of examining the kinase activity or growth potentialof a MARS expressing cell line in the absence of a test compound andcomparing this to the kinase activity or growth potential of a MARSexpressing cell line in the presence of a test compound, wherein andecrease in the kinase activity or growth potential of the MARSexpressing cell line in the presence of a test compound indicates thatsaid compound may be an inhibitor of the biological activity of saidMARS.

Yet another embodiment of the invention is a method of identifying acompound which specifically binds a mutant Bcr-Abl polypeptide; whereinthe Bcr-Abl polypeptide comprises an amino acid substitution that occursin a region of the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1comprising residue D233 through residue T406, the method comprising thesteps of: contacting the mutant Bcr-Abl polypeptide with a test compoundunder conditions favorable to binding; and determining whether the testcompound specifically binds to the mutant Bcr-Abl polypeptide such thata compound which binds to the mutant Bcr-Abl polypeptide can beidentified. The binding of the compound is typically determined by anyone of a wide variety of assays known in the art such as ELISA, RIA,and/or BIAcore assays.

In preferred embodiments, the amino acid substitution in the mutantBcr-Abl polypeptide occurs at residue D233, T243, M244, K245, G249,G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265,V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291,E292, I293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, I314,T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333,E334, A337, V339, L342, M343, A344, I347, A350, M351, E352, E355, K357,N358, F359, I360, L364, E373, N374, K378, V379, A380, D381, F382, T389,T392, T394, A395, H396, A399, P402, or T406. In a specific embodiment ofthe invention, the amino acid substitution is D233H, T243S, M244V,G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R,F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R,D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G,I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S,C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C,Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G,L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K,E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D,K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G,H396K, A399G, P402T or T406A.

A related embodiment of the invention consists of the method describedabove and further comprising determining whether the test compoundinhibits the tyrosine kinase activity of the mutant Bcr-Abl polypeptideby transfecting mammalian cells with a construct encoding the mutantBcr-Abl polypeptide, contacting the mammalian cells with the testcompound; and then monitoring the mammalian cells for the tyrosinekinase activity of the mutant Bcr-Abl polypeptide, wherein an inhibitionin tyrosine kinase activity in the presence of the test compound ascompared to the absence of the test compound identifies the testcompound as an inhibitor of the mutant Bcr-Abl polypeptide. In preferredembodiments of the invention the tyrosine kinase activity of the mutantBcr-Abl polypeptide is measured by examining the phosphotyrosine contentof Crkl.

As illustrated in the Examples below, yet another embodiment of theinvention is a method of determining whether a test compound inhibitsthe tyrosine kinase activity of a mutant Bcr-Abl polypeptide, whereinthe Bcr-Abl polypeptide comprises an amino acid substitution that occursin a region of the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1comprising residue D233 through residue T406, the method comprising thesteps of transfecting mammalian cells (e.g. 293-T cells) with aconstruct encoding the mutant Bcr-Abl polypeptide so that the mutantBcr-Abl polypeptide is expressed by the mammalian cells, contacting themammalian cells with the test compound and then monitoring the mammaliancells for the tyrosine kinase activity of the mutant Bcr-Ablpolypeptide, wherein an inhibition in tyrosine kinase activity in thepresence of the test compound as compared to the absence of the testcompound identifies the test compound as an inhibitor of the mutantBcr-Abl polypeptide. In specific embodiments of the invention, the aminoacid substitution occurs at residue D233, T243, M244, K245, G249, G250,G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268,V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292,I293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, I314, T315,E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334,A337, V339, L342, M343, A344, I347, A350, M351, E352, E355, K357, N358,F359, I360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392,T394, A395, H396, A399, P402, or T406.

Preferably in such methods, the tyrosine kinase activity of the mutantBcr-Abl polypeptide is measured by examining the phosphotyrosine contentof Crkl. Alternatively, the tyrosine kinase activity of the mutantBcr-Abl polypeptide is measured via Western blot analysis using ananti-phosphotyrosine antibody to examine the phosphotyrosine content oflysates of the mammalian cells. These methods can be used to examine awide variety of compounds such as 2-phenylaminopyrimidines orpyrido[2,3-d]pyrimidines.

Typically the amino acid substitution occurs at residue G250, Q252,E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343,A344, M351T, E35, K357, I360, V379 or H396. In certain embodiments ofthe invention, the amino acid substitution does occur at one of theresidues identified in Table IA (e.g. residue T315) but not another ofthe residues identified in Table IA (e.g. residue E255).

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a MARS proteinor a MARS gene or message, respectively. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

EXAMPLES Example 1 Illustrative Materials and Methods for ExaminingBCR-ABL

In an illustrative strategy for examining MARS, our laboratory hasembarked on a large scale sequencing project to identify mutations inthe ABL kinase domain in patients with chronic myeloid leukemia. Apreferred experimental strategy is to use PCR to amplify a region of theBCR-ABL transcript using primers specific to BCR and ABL, subclone thisproduct and sequence at least 10 independent clones in both directions.This strategy allows one to quantify fluctuations in different clonesfrom the same patient over time. Several different groups of patientshave been analyzed in order to determine if the frequency and type ofABL mutation differs with disease stage or prior treatment. These groupsinclude: chronic phase untreated with STI-571 (Gleevec), chronic phasetreated with STI-571, blast crisis untreated with STI-571 and blastcrisis treated with STI-571. Using this strategy we have found over 40such mutations. Typical methodologies are for such protocols areprovided below.

Example 1A Illustrative Methods for Examining BCR-ABL Polynucleotide andPolypeptide Sequences

Blood samples were obtained from consenting patients enrolled inclinical trials at UCLA assessing the efficacy of STI-571 in thetreatment of CML. RNA was extracted using TriReagent or TriAzol. cDNAsynthesis was performed using MMTV reverse transcriptase. Polymerasechain reaction (PCR) was performed using the following primers: CM10(5′-GAAGCTTCTCCCTGACATCCGT-3′) (SEQ ID NO: 6) and 3′ Abl KD(5′-GCCAGGCTCTCGGGTGCAGTCC-3′) (SEQ ID NO: 7). The resultant 1.3 kbfragment was excised from a low melting point agarose gel followingelectrophoresis. A second PCR was performed on the gel-purified 1.3 kbfragment to isolate the kinase domain using the primers 5′ Abl KD(5′-GCGCAACAAGCCCACTGTCTATGG-3′) (SEQ ID NO: 8) and 3′ Abl KD. Theresultant 0.6 kb fragment was ligated into pBluescript II KS+ digestedwith Eco RV. Bacterial transformants were plated on media containingampicillin and X-gal. Ten white colonies per cDNA were inoculated intomedia and miniprep DNA was isolated. Sequencing of each clone wasperformed using M13 universal forward and reverse primers. Because tworounds of amplification were employed, a mutation was considered presentif it was detected on both strands of at least two independent clonesper patient (see FIG. 8). Analysis of the Abl kinase domain from twohealthy blood donors was performed using PCR of the Abl kinase domain,followed by subsequent reamplification to control for the number ofamplification cycles to which patient samples were subjected. Sequenceanalysis of a 0.7 kb portion of Bcr-Abl immediately 5′ to the kinasedomain was performed by amplification of the previously described 1.3 kbfragment using CM10 and 5′ Abl KD reverse complement(5′-CCATAGACAGTGGGCTTGTTGCGC-3′) (SEQ ID NO: 9) followed by ligationinto pBluescript II KS+ as above. The kinase domain of c-Kit wasamplified using the following primers: (5′-TGAGGAGATAAATGGAAACAA-3′)(SEQ ID NO: 10) and (5′-AACTCGTCATCCTCCATGAT-3′) (SEQ ID NO: 11). Tocontrol for the number of cycles used for the Bcr-Abl kinase domain, asecond amplification was performed; the resultant 0.6 kb fragment wassubcloned into pBluescript II KS+ and ten independent colonies weresequenced.

Expression vectors of mutant P210 isoforms were created as follows.Oligonucleotides containing various point mutations were synthesized byGibco/BRL. PSRalphaP210Bcr-Abl was used as the template DNA forsite-directed mutagenesis reactions utilizing the mutantoligonucleotides and the QuikChange mutagenesis kit (Stratagene).Successful mutagenesis was confirmed by sequence analysis of the kinasedomain. Other P210 abl constructs are known in the art (see, e.g. Sun etal., Cancer Res. 2002, 62(11): 3175-3183; Dugray et al., Leukemia 200115(10): 1658-1662; and Heisterkamp et al., Transgenic Res. 1991 1(1):45-53).

293-T cells were co-transfected with mutant P210 expression vectors anda packaging plasmid (Ecopack, kindly provided by R. van Etten). Mediacontaining virus was used to infect Ba/F3 cells. Stable lines wereselected in the presence of G418 and IL-3. Subsequently, IL-3 wasremoved from the media. Expression of Bcr-Abl was document by Westernblot analysis. To determine the biochemical sensitivity of mutant P210isoforms to STI-571, cells were incubated in the presence of STI-571(kindly provided by Novartis, Switzerland) at 0, 0.5, 1, 5, and 10micromoles per liter. After two hours of incubation, cell lysates wereprepared in 1% Triton. Western blot analysis using AB-3 (OncogeneResearch Products) or 4G10 Upstate Biochemicals) was performed. Todetermine the biological sensitivity to STI-571, Ba/F3 cells expressingvarious isoforms of P210 were incubated in the presence of STI-571kindly provided by Novartis, Switzerland) at 0, 0.5, 1, 5, and 10micromoles per liter. After 24 hours of incubation, live cells werequantitated by trypan blue stain exclusion.

Example 1B Illustrative Methods for Examining Discreet Regions inBCR-ABL

In certain contexts, it may be desirable to amplify a specific region inBCR-ABL such as one of the functional domains discussed herein. In thiscontext, a 579 base pair region corresponding to the ATP-binding pocketand the activation loop of the kinase domain of Bcr-Abl was sequenced inthe 9 patients for whom RNA was available at the time of relapse (FIG.4A). Briefly, RNA was extracted from purified peripheral blood or bonemarrow cells with Trireagent-LS (Molecular Research Center, Inc.,Cincinnati, Ohio). 2 mg of total RNA was subjected to RT-PCR using OligodT primers. A 1327-bp cDNA fragment was amplified by PCR with a 5′BCR-specific primer (5′-GAAGCTTCTCCCTGGCATCCGT-3′) (SEQ ID NO: 6) and a3′ ABL-specific primer (5′-GCCAGGCTCTCGGGTGCAGTCC-3′) (SEQ ID NO: 7). Intwo patients, the BCR-ABL fragment could not be amplified; therefore, a579-bp fragment was amplified using an alternative 5′ ABL-specificprimer (5′-GCGCAACAAGCCCACTGTCTATGG-3′) (SEQ ID NO: 8) and the same 3′ABL primer. PCR products were cloned into the pCR2.1 TA cloning vector(Invitrogen, Carlsbad, Calif.). Both strands of a 579-bp region weresequenced with the 5′ ABL primer and M13 forward primer or M13 forwardand reverse primer set for the 1327-bp and the 579-bp fragments,respectively, on an ABI prism 377 automated DNA sequencer (PE AppliedBiosystems, Foster City, Calif.). Sequence analysis was performed usingthe ClustalW alignment algorithm). A single, identical C→T nucleotidechange was detected at ABL nucleotide 944 in six of nine cases examined(FIG. 4A). In all six patients a mixture of wild-type and mutant cDNAclones were found, with the frequency of mutant clones ranging from 17%to 70%. The mutation was found in three of three patients with lymphoiddisease and in three of six patients with myeloid blast crisis. Thepresence of the mutation was confirmed by analysis of genomic DNA (FIG.4A). Briefly, genomic DNA was extracted from purified bone marrow orperipheral blood cells with the QiaAMP Blood Mini Kit (Qiagen, Inc.,Valencia, Calif.). A 361-bp DNA fragment was amplified by PCR with twoprimers (5′-GCAGAGTCAGAATCCTTCAG-3′ (SEQ ID NO: 2) and5′-TTTGTAAAAGGCTGCCCGGC-3′) (SEQ ID NO: 3) which are specific for intronsequences 5′ and 3′ of ABL exon 3, respectively. PCR products werecloned and sequenced. Analysis of RNA or genomic DNA from pre-treatmentsamples failed to provide evidence of the mutation prior to STI-571therapy; however, we cannot rule out the possibility that rare cellsbearing the mutation exist prior to treatment.

Example 2 Illustrative Methods for Measuring of BCR-ABL Kinase ActivityVia the Phosphotyrosine Content of CRKL

Although the enzymatic activity of Bcr-Abl protein is readily measuredin cell lines (e.g. via one of the assays discussed herein below), attimes such assays can be difficult to perform in a reproducible,quantitative fashion with clinical materials because Bcr-Abl is subjectto rapid degradation and dephosphorylation upon cell lysis. In a searchfor alternative measures of Bcr-Abl kinase activity, we found that thephosphotyrosine content of Crkl, an adaptor protein which isspecifically and constitutively phosphorylated by Bcr-Abl in CML cells(see, e.g. J. ten Hoeve et al., Blood 84, 1731 (1994); T. Oda et al., J.Biol. Chem. 269, 22925 (1994); and G. L. Nichols et al., Blood 84, 2912(1994)), could be measured reproducibly and quantitatively in clinicalspecimens. Crkl binds Bcr-Abl directly and plays a functional role inBcr-Abl transformation by linking the kinase signal to downstreameffector pathways (see, e.g. K. Senechal et al., J. Biol. Chem. 271,23255 (1996)). When phosphorylated, Crkl migrates with altered mobilityin SDS-PAGE gels and can be quantified using densitometry. As expected,Crkl phosphorylation in primary CML patient cells was inhibited in adose-dependent manner when exposed to STI-571 and correlated withdephosphorylation of Bcr-Abl (FIG. 1A). This Crkl assay allows for anassessment of the enzymatic activity of Bcr-Abl protein in areproducible, quantitative fashion in clinical materials.

Briefly, cells are lysed in 1% Triton X-100 buffer with protease andphosphatase inhibitors (see, e.g. A. Goga et al., Cell 82, 981 (1995)).Equal amounts of protein, as determined by the BioRad DC protein assay(Bio-Rad Laboratories, Hercules, Calif.), are separated by SDS-PAGE,transferred to nitrocellulose and immunoblotted with phosphotyrosineantibody (4G10, Upstate Biotechnologies, Lake Placid, N.Y.), Ablantibody (pex5, (see, e.g. A. Goga et al., Cell 82, 981 (1995)), β-actinantibody (Sigma Chemicals, St. Louis, Mo.) or Crkl antiserum (Santa CruzBiotechnology, Santa Cruz, Calif.). Immunoreactive bands are visualizedby ECL (Amersham Pharmacia Biotech, Piscataway, N.J.). Several exposuresare obtained to ensure linear range of signal intensity. Optimalexposures are quantified by densitometry using ImageQuant software(Molecular Dynamics, Sunnyvale, Calif.)).

To establish the dynamic range of this assay in patient material, wemeasured Crkl phosphorylation in cells from BCR-ABL-negative individuals(n=4), untreated CML patients (n=4), as well as from patients whoresponded to STI-571 therapy but whose bone marrow cells remained 100%Ph-chromosome-positive (n=8). The mean level of Crkl phosphorylation incells from CML patients prior to STI-571 treatment was 73±13.3% (FIG.1B). At the time of response the mean was 22±9.9% (FIG. 1B), similar tothe mean level of Crkl phosphorylation in cells from BCR-ABL-negativeindividuals (22±6.0%) (see, e.g. M. E. Gorre, C. L. Sawyers). We nextmeasured levels of Crkl phosphorylation in primary leukemia cells from11 patients who responded to STI-571 but subsequently relapsed ontreatment. In these cases, which included one patient with lymphoidblast crisis, three with Ph+ acute lymphoid leukemia, and seven withmyeloid blast crisis, the mean level of Crkl phosphorylation at relapsewas 59±12.5% (FIG. 1C). Anti-phosphotyrosine immunoblot analysis of asubset of these samples confirmed that Bcr-Abl was phosphorylated ontyrosine at relapse (FIG. 1C). Longitudinal analysis of blood or bonemarrow samples obtained from a subset of these patients before andthroughout the course of STI-571 treatment confirmed that Crklphosphorylation fell during the response to treatment, but increased atthe time of relapse (FIG. 1D). Therefore, disease progression inpatients who initially respond to STI-571 is associated with failure tomaintain effective inhibition of Bcr-Abl kinase activity.

Example 3 Illustrative Methods for Examining Amplification of theBCR-ABL Gene in Mammalian Cells

Some CML cell lines that develop resistance to STI-571 after months ofin vitro growth in sub-therapeutic doses of the drug have amplificationof the BCR-ABL gene (see, e.g. E. Weisberg et al., Blood 95, 3498(2000); P. le Coutre et al., Blood 95, 1758 (2000); and F. X. Mahon etal., Blood 96, 1070 (2000)). We performed dual-color fluorescence insitu hybridization (FISH) experiments to determine if BCR-ABL geneamplification could be similarly implicated in STI-571 resistance inhuman clinical samples. Briefly, interphase and metaphase cells wereprepared (see, e.g. E. Abruzzese et al., Cancer Genet. Cytogenet. 105,164 (1998)) and examined using Locus Specific Identifier (LSI) BCR-ABLdual color translocation probe (Vysis, Inc., Downers Grove, Ill.)).Multiple copies of the BCR-ABL gene were detected in interphase nucleiin three (two myeloid blast crisis, one lymphoid blast crisis) of thepatients who relapsed after initially responding to STI-571 (FIG. 3).Further cytogenetic and FISH characterization of metaphase spreads fromthese patients showed a unique inverted duplicate Ph-chromosome withinterstitial amplification of the BCR-ABL fusion gene (FIG. 3C). In onepatient, the inverted duplicate Ph-chromosome could be detected prior tothe initiation of STI-571. In all three cases, additional copies of theaberrant Ph-chromosome were observed as STI-571 treatment continued, aswell as ring chromosomes harboring multiple copies of the BCR-ABL.Patient MB14 was reevaluated by FISH one month after receivingalternative treatment for her leukemia. Strikingly, BCR-ABLamplification was no longer detectable 4 weeks after discontinuation ofSTI-571, raising the possibility that persistent STI-571 administrationmight select for increased copies of the BCR-ABL gene in some patients.

Quantitative PCR analysis of genomic DNA obtained from these threepatients confirmed increased ABL gene copy number at relapse whencompared to a patient without BCR-ABL gene amplification (FIG. 3D).Briefly, genomic DNA was extracted from purified bone marrow orperipheral blood cells with the QiaAMP Blood Mini Kit (Qiagen, Inc.,Valencia, Calif.). 10 ng of total genomic DNA was subjected to real-timePCR analysis with the iCycler iQ system (Bio-Rad Laboratories, Hercules,Calif.). A 361-bp gDNA fragment including ABL exon 3 was amplified usingtwo primers (5′-GCAGAGTCAGAATCCTTCAG-3′ (SEQ ID NO: 2) and5′-=TGTAAAAGGCTGCCCGGC-3′ (SEQ ID NO: 3)) which are specific for intronsequences 5′ and 3′ of ABL exon 3, respectively. A 472-bp gDNA fragmentof glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified usingtwo primers (5′-TTCACCACCATGGAGAAGGC-3′ (SEQ ID NO: 4) and5′-CAGGAAATGAGCTTGACAAA-3′ (SEQ ID NO: 5)) which are specific forsequences in exon 5 and exon 8 of GAPDH, respectively. Fold increase inABL copy number was determined by calculating the difference betweenthreshold cycle numbers of ABL and GAPDH for each sample (DCt). Usingcontrol LB3 as reference sample, DCt from each sample was subtractedfrom DCt of control to determine D(DCt). Fold increase was calculated as2^(−D(DCt)).

Example 4 Art Accepted Methods for Measuring Enzymological andBiological Properties of BCR-ABL Mutants

A variety of assays for measuring the enzymological properties ofprotein kinases such as Abl are known in the art, for example thosedescribed in Konopka et al., Mol Cell Biol. 1985 November;5(11):3116-23; Davis et al., Mol Cell Biol. 1985 January; 5(1):204-13;and Konopka et al., Cell. 1984 July; 37(3):1035-42 the contents of whichare incorporated herein by reference. Using such assays the skilledartisan can measure the enzymological properties of mutant BCR-Ablprotein kinases.

A variety of bioassays for measuring the transforming activities ofprotein kinases such as Abl are known in the art, for example thosedescribed in Lugo et al., Science. 1990 Mar. 2; 247(4946):1079-82; Lugoet al., Mol Cell Biol. 1989 March; 9(3):1263-70; Klucher et al., Blood.1998 May 15; 91(10):3927-34; Renshaw et al., Mol Cell Biol. 1995 March;15(3):1286-93; Sirard et al., Blood. 1994 Mar. 15; 83(6):1575-85;Laneuville et al., Cancer Res. 1994 Mar. 1; 54(5):1360-6; Laneuville etal., Blood. 1992 Oct. 1; 80(7):1788-97; Mandanas et al., Leukemia. 1992August; 6(8):796-800; and Laneuville et al., Oncogene. 1991 February;6(2):275-82 the contents of which are incorporated herein by reference.Using such assays the skilled artisan can measure the phenotype ofmutant BCR-Abl protein kinases.

Using protocols known in the art we have shown that T315I and E255K bothretain potent kinase activity and can confer growth factor independencein BaF3 murine hematopoietic cells. This mutant is resistant toinhibition by STI-571 in kinase assays and in growth assays. Othermutants can be similarly studied using such analyses.

Example 5 Additional Illustrative Analytical Schemes for Characterizingthe Functional Importance of BCR-ABL Mutations

In addition to the methods described above, skilled artisans canundertake additional analyses of one or more BCR-ABL mutants such asthose identified in Table I. For example, typical illustrativealgorithms such as those whose parameters are outlined below can be usedto characterize the clinical importance of the various mutations foundin the kinase domain.

In a first illustrative method, one can examine samples from the samepatient obtained at different times during their disease progression.Clones which become dominant over time may be presumed to have a growthadvantage. This advantage could, for example be a consequence ofincreased potency of the BCR-ABL oncogene or resistance to a drugtreatment such as STI-571 (as demonstrated by the T315I mutation). Inaddition, mutations which appear more commonly can be given priority.

In a second illustrative method, one can examine the location of themutation in the context of the crystal structure of the ABL kinasedomain (which has been solved bound to STI-571). This structure allowsone to postulate whether the mutation might interfere with theanti-leukemia activity of STI-571. Based on this analysis, one canprioritize mutations for direct experimental analysis of ABL kinaseactivity, leukemogenicity and level of inhibition by STI-571.

In yet another illustrative method, one can engineer selected mutationsinto wild-type BCR-ABL cDNA to create a mutant allele whoseenzymological and biological properties can be examined directly (see,e.g. Example 1 above). Enzymology can be performed by measuring tyrosinekinase activity in vitro or in cells using standard assays known in theart. Biological activity can be measured using standard oncogenetransformation assays using growth factor dependent hematopoietic celllines or primary mouse bone marrow cells. Resistance to STI-571 can bemeasured using kinase assays and transformation assays.

Example 6 Use of Information Regarding BCR-ABL Domains andCrystallographic Analysis to Characterize BCR-ABL Mutations

As the certain domains within BCR-ABL have been characterized and thecrystal structure of this protein has been elucidated, this informationcan be used in conjunction with the disclosure provided herein tocharacterize MARS such those shown in Table I and to illustrate theirrole in resistance to inhibition of tyrosine kinase activity by STI-571.For example, from the initial inspection of these mutations in thecontext of the ABL crystal structure, one can categorize the mutants,for example in the following groups:

1. Helix C mutations (e.g. amino acid residue positions 304, 278):Helix C is a key regulatory helix in the kinase V304D, V304A. These arelocated at the interface with helix C; M278K, M278L: Surface exposedmethionine is disordered (borders helix C). The functional significanceof mutations found within this region or proximal to this region (in amanner that can perturb the normal function of this region), aresupported by references which characterize this aspect of BCR-ABL.2. P loop mutations (e.g. amino acid residue positions 253, 252, 250):The P loop is the phosphate binding loop whose conformation is thoughtto be induced by STI-571. These mutations could prevent the requiredconformation of the loop to accommodate STI-571. Interestingly, we havefound no mutations in the Gly motifs in the P loop (249, 251 and 254).These are highly conserved across other kinases (so calledGly-X-Gly-X-X-Gly motif) and presumably are essential for kinasefunction. We do have examples of mutations in each of the X positions inthe P loop. The functional significance of mutations found within thisregion or proximal to this region (in a manner that can perturb thenormal function of this region), are supported by references whichcharacterize this aspect of BCR-ABL.

Y253F: Directly stacks up against STI-571. —OH makes a tight H-bond withCL (or H2O). Others: Q252H, Q252L, Q252R, G250E, E255K.

3. Residues which directly interact with STI-571 (e.g. amino acidresidue positions 315, 351, 355, 317, 290): The functional significanceof these residues or proximal to these residues (in a manner that canperturb the normal function of this region), are supported by referenceswhich characterize this aspect of BCR-ABL.

M351T: van der Waal interactions with His 361 which in turn interactsdirectly with STI-571 piperazine group. Thr mutation could disrupt thepacking here and weaken interaction with STI-571. Interestingly, thismutation may not affect compound 15 binding (the one originallycrystallized with Abl) since it has no piperazine group.

E355G: at the end of the helix that precedes the catalytic loop, whichinteracts with the piperazine group of STI-571. Mutating to a Gly couldmake this region more flexible and weaken STI binding. Again Compound 15should be less affected by this mutation.

F317L: directly stacks against STI-571. Leu mutation could weakenSTI-571 binding.

M290T, M290V: makes direct van der Waal interactions with STI-571.Mutation to either T or V would weaken STI-571 binding.

4) Activation loop mutations (e.g. amino acid residue positions 396).The functional significance of mutations found within this region orproximal to this region (in a manner that can perturb the normalfunction of this region), are supported by references which characterizethis aspect of BCR-ABL.

H396K, H396R: disordered part of the activation loop.

Example 7 BCR-ABL Point Mutants Isolated from Patients withSTI571-Resistant Chronic Myeloid Leukemia Remain Sensitive to Inhibitorsof the BCR-ABL Chaperone Heat Shock Protein 90

Clinical resistance to STI571 (Gleevec/imatinib mesylate) is commonlyobserved in patients with advanced Philadelphia chromosome-positive(Ph⁺) leukemias. Acquired resistance is typically associated withreactivation of BCR-ABL due to kinase domain mutations or geneamplification, indicating that BCR-ABL remains a viable target forinhibition in these patients. Strategies for overcoming resistance canbe envisioned through exploitation of other molecular features of theBCR-ABL protein, such as its dependence on the molecular chaperone heatshock protein 90 (Hsp90). To determine whether inhibition of Hsp90 couldinduce degradation of STI571-resistant, mutant BCR-ABL proteins,hematopoietic cells expressing two mutant BCR-ABL proteins found inSTI571-resistant patients (T315I and E255K) were examined forsensitivity to geldanamycin and 17-AAG. Both compounds induced thedegradation of wild-type and mutant BCR-ABL and inhibited cell growth,with a trend indicating more potent activity against mutant BCR-ABLproteins. These data support clinical investigations of 17-AAG inSTI571-resistant Ph-positive leukemias.

Strategies for overcoming resistance associated with kinase domainmutations will likely require targeting other molecular features of theBCR-ABL protein. Heat shock protein 90 (Hsp90) is a molecular chaperonewhich affects the stability and function of multiple oncogenic proteinsincluding BCR-ABL (An W G et al., Cell Growth Differ. 2000; 11:355-360;Shiotsu et al., Blood. 2000; 96:2284-2291). Geldanamycin (GA) is abenzoquinone ansamycin which specifically inhibits Hsp90 bycompetitively binding to an ATP-binding pocket in the amino-terminus ofHsp90 (Prodromou et al., Cell. 1997; 90:65-75; Stebbins et al., Cell.1997; 89:239-250; Grenert et al., 1997; 272:23843-23850). Disruption ofHsp90 function by geldanamycin or its less toxic analogue,17-allylaminogeldanamycin (17-AAG), in BCR-ABL-expressing leukemia cellshas been shown to induce BCR-ABL protein degradation and suppress cellproliferation (An W G et al., Cell Growth Differ. 2000; 11:355-360;Blagosklonny M V, et al., Leukemia. 2001; 15:1537-1543; Nimmanapalli R,et al., Cancer Res. 2001; 61:1799-1804). 17-AAG is currently in phase Iclinical trials.

To determine whether inhibition of Hsp90 could induce degradation ofSTI571-resistant, mutant BCR-ABL proteins, hematopoietic cellsexpressing two mutant BCR-ABL proteins found in STI571-resistantpatients (T315I and E255K) were derived and tested for sensitivity togeldanamycin and 17-AAG. We found that both compounds induced thedegradation of wild-type and mutant BCR-ABL proteins as well asinhibited cell growth. The data also suggest a trend indicating agreater potency against mutant BCR-ABL proteins. These results provide arationale for the use of 17-AAG in the clinical setting ofSTI1571-resistant Ph-positive leukemia.

Chemicals. Stock solutions of GA (Sigma), 17-AAG (NSC 330507, NationalCancer Institute), and STI571 (Novartis) were prepared as 10 mMdimethylsulfoxide solutions and stored at −20° C.Plasmids and cell lines. Full-length P210 T315I and P210 E255K BCR-ABLin pBluescript (Stratagene) were generated using site-directedmutagenesis and confirmed by sequencing as described previously (Gorreet al., Science. 2001; 293:876-880). Wild-type and mutant P210 BCR-ABLwere subsequently subcloned into the EcoRI site of pMSCVpuro (Clontech)for retrovirus generation. Ecotropic retroviruses were generated bycotransfection of pMSCVpuro DNA and Ecopac retroviral packaging vector(kindly provided by R. Van Etten) into 293T cells using the CaCl₂ method(Muller A J, et al., Mol. Cell. Biol. 1991; 11:1785-1792). The murinehematopoietic cell line Ba/F3 was maintained in RPMI1640 supplementedwith 10% fetal bovine serum, L-glutamine, and 1 ng/ml of recombinantmurine IL-3 (R&D). Ba/F3 populations with stable BCR-ABL expression werederived by retroviral infection of Ba/F3 cells in the presence of IL-3,and subsequent selection by puromycin. IL-3-independentBCR-ABL-expressing cells were derived by culturing in IL-3-free media atlow densities in 96-well tissue culture plates. MultipleIL-3-independent populations were assayed for comparable BCR-ABL proteinexpression by western blot.

In vitro drug exposure assays. Cells were cultured in 24-well plates at2×10⁵ cells/ml in growth media (plus IL-3 for parental cells) with GA,17-AAG, or STI571 for 24 or 48 hours. Subsequent analyses of protein bywestern blot or cell viability by trypan blue dye exclusion were done aspreviously described (Gorre et al., Science. 2001; 293:876-880; Goga A,et al., Cell. 1995; 82:981-988).

Results

Previous studies have shown that the Hsp90 inhibitors GA and itsderivative, 17-AAG, disrupt Hsp90 function and induce BCR-ABL proteindegradation (An W G et al., Cell Growth Differ. 2000; 11:355-360;Blagosklonny M V, et al., Leukemia. 2001; 15:1537-1543; Nimmanapalli R,et al., Cancer Res. 2001; 61:1799-1804). To determine whether GA cansimilarly cause the degradation of BCR-ABL proteins carryingSTI571-resistant point mutations, populations of interleukin-3 (IL-3)dependent Ba/F3 murine hematopoietic cells were engineered to expresseither wild-type, T315I, or E255K P210 BCR-ABL and exposed to varyingconcentrations of inhibitor. Consistent with previous reports, bothmutant BCR-ABL alleles rendered the cells independent of IL-3, and cellsexpressing either mutant contained high levels of phosphotyrosine onBCR-ABL and other substrate proteins (Gorre et al., Science. 2001;293:876-880; von Bubnoff et al., Lancet. 2002; 359:487-491). Westernblot analyses using ABL-specific antibodies demonstrated that GA causedBCR-ABL protein levels to decrease significantly in cells expressingwild-type BCR-ABL after treatment for 24 hours at a dose of 1.0 μM, asexpected (An W G et al., Cell Growth Differ. 2000; 11:355-360;Blagosklonny M V, et al., Leukemia. 2001; 15:1537-1543; Nimmanapalli R,et al., Cancer Res. 2001; 61:1799-1804). BCR-ABL protein was alsodegraded in cells expressing either T315I or E255K BCR-ABL, but thisdegradation occurred at a lower GA concentration (0.5 μM) (FIG. 7A).This apparently enhanced degradation of the two mutant BCR-ABL proteinswas specific because degradation of another Hsp90 client protein, RAF-1,was comparable in all cells tested. These data suggest that GA may havegreater potency against mutant BCR-ABL proteins compared to wild-type.

We next tested 17-AAG—a GA derivative currently in phase I clinicaltrials—for its ability to induce BCR-ABL protein degradation in the sameBa/F3 cell lines. Western blot analyses of lysates from cells culturedin 17-AAG showed a similar trend to that seen with GA. Wild-type BCR-ABLprotein levels fell gradually after 24 hour exposure to 0.5-1.0 μM17-AAG. Although BCR-ABL protein levels in both the T315I and E255KBCR-ABL-expressing cells began to decline at a similar concentration of17-AAG as wild-type BCR-ABL (0.5 μM), the magnitude of decrease was moredramatic in cells expressing the BCR-ABL mutants. Virtually no BCR-ABLprotein was detectable at 1.0 μM of 17-AAG for both mutants (FIG. 7B).This trend was confirmed when we assessed the effect of 17-AAG ondownstream BCR-ABL signaling by measuring the phosphorylation status ofCRKL, a direct BCR-ABL substrate with functional relevance in CML(Nichols et al., Blood. 1994; 84:2912-2918; Oda et al., J. Biol. Chem.1994; 269:22925-22928; Senechal et al., J. Biol. Chem. 1996;271:23255-23261; ten Hoeve J et al., Blood. 1994; 84:1731-1736). Westernblot analysis using CRKL-specific antisera on lysates from cellsincubated in the presence of increasing concentrations of STI571confirmed that the BCR-ABL mutants conferred resistance to STI571 (FIG.7E). CRKL western blot analysis on lysates from 17-AAG-treated cellsrevealed that lower doses of 17-AAG were needed to inhibit BCR-ABLactivity in cells expressing the BCR-ABL mutants when compared towild-type BCR-ABL (FIG. 7C,D). Significant changes in CRKLphosphorylation were not observed in wild-type BCR-ABL-expressing cellsuntil a 17-AAG concentration of 5.0 μM was reached, whereas CRKLphosphorylation in T315I and E255K BCR-ABL-expressing cells wassignificantly inhibited at 0.5 μM of drug (FIG. 7C,D). While 17-AAG mayaffect another kinase which plays a role in CRKL phosphorylation inthese cells, the fact that 17-AAG also reduced the level of BCR-ABLprotein, together with previously published data showing thatconstitutively elevated CRKL phosphorylation is relatively specific forCML (Nichols et al., Blood. 1994; 84:2912-2918), provides strongevidence that BCR-ABL is the target.

Previous studies have also shown that GA and 17-AAG inhibit growth andinduce apoptosis of BCR-ABL-positive leukemic cell lines (Blagosklonny MV, et al., Leukemia. 2001; 15:1537-1543; Nimmanapalli R, et al., CancerRes. 2001; 61:1799-1804). To determine whether GA could inhibit growthin cells expressing STI571-resistant BCR-ABL mutants, Ba/F3 cellstransformed by wild-type, T315I, and E255K BCR-ABL were cultured in arange of GA concentrations. Trypan blue dye exclusion assessments ofviability and corresponding IC₅₀ calculations indicated that the growthof all three BCR-ABL-positive cell lines was inhibited by GA at lowerdoses when compared to BCR-ABL-negative parental cells (Table III). Theenhanced sensitivity of the STI-571 resistant BCR-ABL mutants comparedto wild-type BCR-ABL observed in the biochemical analyses was alsorecapitulated in the growth inhibition assays. Similar results wereobserved with 17-AAG-treated cells. All BCR-ABL-expressing cells weremore sensitive to 17-AAG than Ba/F3 parental cells, and theSTI571-resistant BCR-ABL-expressing cells again displaying a heightenedsensitivity to inhibition compared to wild-type BCR-ABL-expressing cells(Table III).

In summary, targeted inhibition of Hsp90 with either GA or 17-AAGinduced the degradation of wild-type BCR-ABL and two STI571-resistantBCR-ABL mutants T315I and E255K. Both compounds also inhibited thegrowth of hematopoietic cells transformed by wild-type and mutantBCR-ABL. The results also suggest that the STI571-resistant mutants aremore sensitive to Hsp90 inhibition than wild-type BCR-ABL. One potentialexplanation could be that these two mutant proteins are less stable thanwild-type BCR-ABL, and therefore more dependent on molecular chaperones.A better understanding of the variables that determine the relativedependence of client proteins on Hsp90 function is required to fullyevaluate this question. Nevertheless, these data provide support forclinical investigations of 17-AAG in STI571-resistant Ph-positiveleukemia.

Example 8 Identification of a Novel Pyridopyrimidine Inhibitor of ABLKinase that is a Picomolar Inhibitor of BCR-ABL Driven K562 Cells and isEffective Against STI571-Resistant BCR-ABL Mutants

Inhibition of the constitutively active Bcr-abl tyrosine kinase (TK) bySTI571 has proven to be a highly effective treatment for chronicmyelogenous leukemia (CML). However STI571 is only transiently effectivein blast crisis and drug resistance emerges by amplification of ordevelopment of mutational changes in Bcr-abl. As described in thisexample, we have screened a family of TK inhibitors of thepyrido[2,3-d]pyrimidine class, unrelated to STI571, and describe here acompound with substantial activity against STI-resistant mutant Bcr-ablproteins. This compound, PD166326, is a dual specificity TK inhibitorand inhibits src and abl in vitro with IC₅₀s of 6 and 8 nM respectively.PD166326 inhibits the growth of K562 cells with IC₅₀ of 300 picomolar,leading to apoptotic G1 arrest, while non-Bcr-abl cell types requiremore than 1000 times higher concentrations. We tested the effects ofPD166326 on two of the clinically observed Bcr-abl mutants. The T315Imutation within the ATP-binding pocket reduces the affinity of STI571for this pocket while the structural basis for resistance of the E255Kmutation is currently unknown. PD166326 potently inhibits the E255Kmutant Bcr-abl protein and the growth of Bcr-abIE255K driven cells. TheT315I mutant Bcr-abl protein is resistant to PD166326, however thegrowth of Bcr-ablT315I driven cells is partially sensitive to thiscompound, likely through the inhibition of Bcr-abl effector pathways.These findings show that tyrosine kinase drug resistance is astructure-specific phenomenon and can be overcome by TK inhibitors ofother structural classes, suggesting new approaches for futureanti-cancer drug development. PD166326 is a prototype of a newgeneration of anti-Bcr-abl compounds with picomolar potency andsubstantial activity against STI571-resistant mutants.

Cell Culture and Growth Assays

Cell were cultured in RPMI medium supplemented with 100 U/ml penicillin,100 μg/ml streptomycin, 4 mM glutamine, 10% heat inactivated fetalbovine serum and incubated at 37 C in 5% CO₂. For growth assays, cellswere seeded in 12-well clusters at 10-20,000 cells per well. Cells wereplaced in media containing various concentrations of the drugs withvehicle (DMSO) never contributing more than 0.1%. After 4-7 days, cellswere counted using a coulter counter. All experiments were performed induplicate and results averaged. PD166326 was stored in a 10 mg/ml DMSOsolution and stored at −70C. The derivation and chemical structure ofPD166326 has been previously published (see e.g. Kraker et al.,Biochemical Pharmacology 60, 885-898. 2000).

Cell Cycle Assays

Cells were treated with indicated concentrations of PD166326 or vehicle(DMSO) for the indicated times. For synchronization, cells wereincubated in media containing 5 ug/ml aphidicolin for 24 hours, washedtwice in PBS, and replaced in growth media. At the time of harvest,cells were washed once in PBS and cell nuclei prepared by the method ofNusse (see e.g. Nusse et al., Cytometry. 1990; 11:813-821) and cellcycle distribution determined by flow cytometric analysis of DNA contentusing red fluorescence of 488 nm excited ethidium bromide stainednuclei.

Protein Extraction and Western Blotting

Cells were washed in PBS once and lysed in modified RIPA buffer (10 mMNa phosphate pH 7.2, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% NP-40,1% Na deoxycholate, 1 mM Na Vanadate, and protease inhibitors). 50 ug oftotal cellular protein was separated by SDS-PAGE, transferred tomembrane, and immunoblotted using antibodies to phosphotyrosine(SantaCruz), c-abl (8E9), and phospho-Hck (SantaCruz), MAP kinase(SantaCruz) and phospho-MAP kinase (Promega).

In Vitro Kinase Assay

C-abl kinase assays were performed using purified recombinant c-abl andpeptide substrate (New England Biolabs). Kinase assays were performed in50 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 1 mM ethylene glycol bis-aminoethylether tetraacetic acid (EGTA), 2 mM dithiothreitol (DTI), 0.2% triton-X,100 uM ATP, 40 uM peptide substrate, in 100 ul reaction volumescontaining 50 units c-abl enzyme and 10 uCi [³²P] γ-ATP. Reactions wereallowed to proceed for 10 minutes at 30 C and stopped by addition ofEDTA and boiling. Reaction products were spotted on phosphocellulosepaper, washed several times with phosphoric acid, then acetone, andcounted in scintillation fluid. Pilot experiments were initiallyperformed to establish that these reaction conditions were in linearrange.

Bcr-Abl was immune precipitated from cell lysates of K562 cellsmaintained in log-phase culture conditions. Complexes were collected onprotein A-sepharose and washed three times in lysis buffer and twice inabl kinase buffer (50 mM tris pH 8.0, 10 mM MgCl₂, 1 mM DTT, and 2 mMp-nitrophenylphosphate, and 2 mM ATP; New England Biolabs Buffer andprotocol). Kinase assays were performed with 10 mM [γ-32P] ATP/samplefor 15-60 minutes at 30 C in the presence or absence of indicatedconcentrations of drug. The immune complexes were pre-incubated with thedrug for 10 minutes at 4 C prior to addition of labelled ATP andinitiation of the reaction at 30 C. The reaction was stopped by theaddition of SDS-PAGE sample buffer and heated at 100 C for 10 minutes.Proteins were separated on 7.5% SDS-polyacrylamide gels and gels weredried under vacuum and phosphorylation was visualized by autoradiographyon x-ray film.

Results

In screening a compound library for inhibitors of c-src tyrosine kinaseactivity, a number of pyrido[2,3-d]pyrimidines were previously describedthat are ATP-competitive inhibitors of c-src with IC₅₀ values <20 nM andvarying degrees of selectivity for c-src (see e.g. Kraker et al.,Biochemical Pharmacology 60, 885-898. 2000). We screened this group ofcompounds for activity against c-abl using purified recombinant c-abland peptide substrate in in vitro kinase assays. The most potentcompound was PD166326 with an IC₅₀ of 8 nM (against c-abl) and 6 nM(against src). The src family kinase Lck is inhibited with IC₅₀<5 nM.This compound also has activity against basic-FGF, PDGF, and EGFreceptor tyrosine kinases in vitro with IC₅₀s of 62, 139, and 80 nMrespectively. PD166326 shows no significant activity against JNKkinases, cyclic AMP-dependent protein kinase (PKA), PKB-β, PKC-α,rho-dependent protein kinase, casein kinase-2, and phosphorylase kinase.In comparison with PD166326, STI571 is a weaker inhibitor of Abl invitro with an IC₅₀=50 nM. PD166326 also inhibits Bcr-abl kinase in vitrowith IC₅₀=1 nM.

PD166326 also inhibits Bcr-abl activity in cells as determined byWestern blot analysis of Bcr-abl autophosphorylation in K562 cells. Inthese cells Bcr-abl autophosphorylation is inhibited with IC₅₀ of 1 nMcompared with 100 nM for STI571. Bcr-abl autophosphorylation correlateswith Bcr-abl signaling activity as shown by the parallel decline of MAPkinase activity with inhibition of Bcr-abl in these assays.

The biologic activity and potency of PD166326 was initially evaluated incell growth assays using K562 cells. This compound inhibits K562 cellgrowth with IC₅₀=0.3 nM. Other Bcr-abl driven cell lines are alsoextremely sensitive to PD166326 with IC₅₀s of 0.8 and 6 nM (seeM07-p210^(bcr-abl) and BaF3-p210^(bcr-abl)). The potent biologicactivity of PD166326 is highly specific for Bcr-abl-driven cells asadditional hematopoetic and epithelial cell lines are only inhibited at2 to 3 logs higher concentrations and IC₅₀s in the 0.8-2 uM range.

Further analysis reveals that PD166326 inhibits cell proliferationspecifically in the G1 phase of the cell cycle. At concentrations thatfully inhibit the growth of Bcr-abl positive cells but not other celltypes, PD166326 leads to accumulation of cells in the G1 phaseaccompanied by a significant increase in the number of apoptotic cells.Additional phases of the cell cycle are not affected by this compound asshown by experiments with synchronized cells. K562 cells weresynchronized at the G1/S boundary with aphidicolin and released intoPD166326 or vehicle and cell cycle progression studied over thefollowing 24 hours. These data show that PD166326 treatment does notinterfere with progression through the S, G2 or mitotic phases of thecell cycle, but PD166326 treated cells are unable to exit the G1 phase.Similar experiments with nocodazole-synchronized cells also confirm thatPD166326 blocks G1 progression. The inhibition of G1 progression andinduction of apoptosis in K562 cells are similar to the effectspreviously reported for STI571 (see e.g. Dan et al., Cell Death &Differentiation. 1998; 5:710-715). These data show that PD166326 is apotent inhibitor of Bcr-abl kinase activity and inhibits Bcr-abl drivencell growth through inhibition of G1 progression leading to apoptoticcell death.

Resistance to STI571 treatment is associated with mutations in theBcr-abl oncoprotein that render it refractory to STI571 inhibition (seee.g. Gorre et al., Science. 2001; 293:876-880). Because PD166326inhibits both Src and Abl whereas STI571 only inhibits Abl, it may bindBcr-abl differently than STI571. This difference raises the possibilitythat it may be effective against some mutant Bcr-abl proteins. Wecompared the activities of PD166326 and STI571 against two such mutantBcr-abl proteins derived from patients who have relapsed on STI571therapy. The T315I mutation is frequently seen in relapsed patients andeliminates a critical Threonine residue within the ATP binding pocket ofAbl and greatly reduces the binding affinity of STI571. The E255Kmutation also lies within a region of Bcr-abl commonly mutated inrelapsed patients, however the structural basis for STI571 resistanceconferred by mutations in this region is not currently understood. BaF3mouse hematopoietic cell lines were stably transfected with either thewild-type p210^(bcr-abl) cDNA or the T315I or E255K mutant versions aspreviously described (see e.g. Gorre et al., Science. 2001;293:876-880). Expression of Bcr-abl renders BaF3 cells IL-3 independentwhile control cells transfected with vector alone require IL-3 forgrowth. Although STI571 inhibits the wild-type p210^(bcr-abl) cells withIC₅₀=500 nM, the T315I and E255K mutant p210^(bcr-abl) cells are highlyresistant. However resistance to STI571 does not appear to confercross-resistance to PD166326. PD166326 inhibits the autophosphorylationof p210^(Bcr-ablE255K) in vivo as effectively as the autophosphorylationof the wild type p210^(Bcr-abl), while this mutant is highly resistantto inhibition by STI571. However, the p210^(Bcr-ablT315I) mutant isresistant to PD166326 as it is to STI571. This is not surprising,considering the critical role of Thr³¹⁵ within the ATP binding pocket.

To determine whether cell growth sensitivity to PD166326 correlates withinhibition of the mutant Bcr-abl oncoproteins, we also determined thesensitivity of the BaF3 cells driven by the wild type and mutant Bcr-ablproteins. BaF3^(p210Bcr-abl) cells are very sensitive to PD166326(IC₅₀=6 nM) and the E255K mutant p210^(bcr-abl) cells remain relativelysensitive to this compound (IC₅₀=15 nM). The effective inhibition ofp210^(E255Kbcr-abl) activity at dose ranges that inhibit the growth ofthese cells is further evidence that STI571-resistant leukemic cells aredriven by persistent activity of the mutated Bcr-abl oncoprotein. Incomparison, the T315I mutant cells are partially resistant to PD166326,although not fully resistant. PD166326 inhibits BaF3^(p210T315I) cellswith IC₅₀ of 150 nM. Although this is 25 fold weaker than the inhibitionof the wild-type BaF3^(p210) cells, it may still be of therapeutic valuesince it is 8-fold more potent than the inhibition of the BaF3-vectorcontrols and non-Bcr-abl driven cells. Although PD166326 inhibits thegrowth of BaF3p210^(Bcr-ablT315I) cells with IC₅₀ of 150 nM, it fails toinhibit the autophosphorylation of the T351I Bcr-abl mutant at doses upto 1 uM, suggesting that its anti-proliferative effects are mediated inpart through mechanisms other than the inhibition of Bcr-abl.

PD166326 is also active against src kinases and its anti-leukemiceffects may be in part related to its inhibition of the src kinases Hckand Lyn which function downstream of Bcr-abl. The src kinases Hck andLyn are activated by Bcr-abl and may mediate some of the transformingfunctions of Bcr-abl. Phosphorylation of tyr⁴¹⁶ in the catalytic domainis required for activation of src kinases, although the mechanism bywhich Bcr-abl activates Hck and Lyn is not understood. Inhibition ofBcr-abl by STI571 results in a parallel inhibition of Hck activation inK562 cells. In these cells PD166326 also inhibits Bcr-abl and Hckactivation although at 100 fold lower doses than seen with STI571. Hckis also activated by mutant forms of Bcr-abl and in the mutantBaF3p210^(Bcr-ablE255K) cells, PD166326 inhibits Hck activation and thiscorrelates with the observed inhibition of Bcr-abl^(E255K)autophosphorylation and inhibition of cell growth. In contrast, theactivation of Hck by the Bcr-abl^(T315I) mutant is not inhibited byPD166326 and this correlates with the observed resistance ofBcr-abl^(T315I) activity to PD166326. However despite failure to inhibitBcr-abl activity and the consequent activation of Hck, PD166326 inhibitsthe growth of BaF3p210^(Bcr-ablT315I) cells with IC₅₀ of 150 nM, likelythrough additional mechanisms.

Although STI571 has revolutionized the treatment of CML, the problem ofTK drug resistance is now emerging as a clinical reality. Resistance toSTI571 appears to have a structural basis and newer TK inhibitors mayalso be susceptible to similar mechanisms of resistance. However TKinhibitors of a different structural class may have more favorablebinding characteristics. Dorsey et al initially reported that asrc-selective TK inhibitor of the pyrido[2,3-d]pyrimidine class hassubstantial activity against Bcr-abl kinase (see e.g. Dorsey et al.,Cancer Research. 2000; 60:3127-3131). We have extended this finding byscreening a family of src-selective pyrido[2,3-d]pyrimidines andidentified a compound with the most potent activity against abl kinase.Here we report the characterization of this compound, PD166326, a noveldual specificity TK inhibitor that is more than 100 fold more potentthan STI571 in vivo and inhibits K562 cells with IC₅₀ of 300 picomolar.It is unlikely that the potent growth inhibitory activities of PD166326are related to non-specific activities since the potency of thiscompound appears to be specific for cell types driven by Bcr-abl kinase.While Bcr-abl-driven cells are inhibited with IC₅₀s in the 0.3-6 nMrange, other cell types including the hematopoietic cells BaF3 and 32Das well as epithelial cancer cells including MCF-7 cells and MDA-MB-468cells, which are driven by EGFR overactivity, are inhibited with IC₅₀sin the 0.8-2 uM range (table 2). The micromolar activity of PD166326against the growth of non-Bcr-abl driven cells is most likely mediatedthrough inhibition of additional cellular targets since unlike Bcr-ablpositive cells, the growth of Bcr-abl negative cells is inhibited duringthe S phase of the cell cycle. The picomolar potency and cellularselectivity of PD166326 are significantly superior to STI571 in vitro.

Since Bcr-abl signaling is known to involve the src family kinases Hckand Lyn, and since PD166326 is also a potent inhibitor of src familykinases, it is plausible that the biologic potency of this compound isrelated to dual inhibition of these two functionally related tyrosinekinases. Hck associates with and phosphorylates Bcr-abl on Tyr 177leading to recruitment of Grb2/Sos and activation of the Ras pathway(see e.g. Warmuth et al., Journal of Biological Chemistry. 1997;272:33260-33270). Kinase-defective Hck mutants suppress Bcr-abl inducedtransformation suggesting that Hck-mediated signaling is essential forthe transforming activity of Bcr-abl (see e.g. Lionberger et al.,Journal of Biological Chemistry. 2000; 275:18581-18585). The role of Lynin Bcr-abl signaling is less well studied. However Lyn activity is alsoelevated in acute myeloid leukemia cell lines and in these cellsinhibition of Lyn expression using anti-sense molecules leads todecreased proliferative activity and inhibition of Lyn kinase activityusing src family selective pharmacologic inhibitors leads to potentinhibition of cell growth and colony formation (see e.g. Roginskaya etal., Leukemia. 1999; 13:855-861). It is also possible that the potencyof PD166326 is mediated through the inhibition of other, yetundiscovered cellular proteins, and our data does not exclude thispossibility. However the role of currently unknown cellular targets inmediating the growth inhibitory effects of this compound in Bcr-abldriven cells is difficult to know until such candidate targets areidentified and studied.

Since relapse on STI571 is associated with mutations in Bcr-abl thatalter the binding of STI571, understanding the nature of the STI571interaction with Abl is of fundamental importance in order to overcomedrug resistance. The crystal structure of a variant STI571 in complexwith the catalytic domain of Abl was recently solved by Schindler et al(see e.g. Schindler et al., Science. 2000; 289:1938-1942). STI bindswithin the ATP binding pocket of Abl in its inactive conformation. Thisinteraction is critically affected by the conformation of the Ablactivation loop. When phosphorylated, this activation loop favors anopen and activating conformation which, by virtue of its amino-terminalanchor, interferes with STI571 binding to the ATP-binding pocket.Consistent with this model, the binding of STI571 is selective for theinactive conformation of Abl, and this compound is unable to inhibit thecatalytic activity of active phosphorylated Abl (see e.g. Schindler etal., Science. 2000; 289:1938-1942). The broader activity of PD166326,including activity against src kinases suggests that unlike STI571, itmay not bind selectively to the inactive conformation of Abl, since inits active conformation, Abl bears considerable structural homology tothe src kinases (see e.g. Schindler et al., Science. 2000;289:1938-1942). While selectivity for the inactive conformation ispostulated to confer a high degree of molecular specificity to STI571,this may be at the price of potency. PD166326 may be binding to bothinactive and active conformations of Abl leading to the more effectiveinhibition of overall enzyme activity that we see in vitro. In addition,phosphorylation of the activation loop of Abl is catalyzed by the srcfamily kinase Hck in Bcr-abl transformed cells. Since PD166326 alsoinhibits Hck, this may prevent phosphorylation of the activation loop,destabilizing the Abl active conformation. This allosteric mechanism inaddition to the direct binding of PD166326 to the ATP-binding pocketcould provide dual mechanisms for its inhibition of Abl activation andprovide the basis for its increased potency. Validation of thesehypotheses awaits crystallographic studies of PD166326 bound to Abl.

PD166326 is non-cross-resistant with STI571 and has substantial activityagainst the T315I and E255K STI571-resistant Bcr-abl mutants. Thisfinding has important implications for the future design and use of TKinhibitors of all kinds, since it is the first report showing thatTK-inhibitor resistance can be overcome by another TK-inhibitor of adifferent structural class. It is difficult to speculate on whether thedevelopment of resistance to PD166326 will be just as likely as withSTI571, but since these compounds are structurally unrelated, resistanceto PD166326 will likely involve a different structural basis thanresistance to STI571. This distinction creates the opportunity forstrategies to prevent or overcome resistance such as sequential orcombination therapies. However understanding drug sensitivity andresistance is of fundamental importance in this regard.

While additional studies will elucidate the exact structural andcellular basis underlying STI571 resistance and PD166326 sensitivity,existing data explains our findings. A number of amino acid residuesmediate the binding of STI571 within the ATP-binding pocket, and amongthese, Thr³¹⁵ is critical for hydrogen bond formation with the drug (seee.g. Schindler et al., Science. 2000; 289:1938-1942). The T315Imutation, seen in STI571-resistant CML, precludes hydrogen bonding withSTI571 and results in a steric clash due to the extra hydrocarbon groupin Ile (see e.g. Gorre et al., Science. 2001; 293:876-880). LikewisePD166326 does not inhibit the activity of Bcr-abl^(T315I) in vivosuggesting that Thr³¹⁵ is also important for its binding within the ATPpocket of abl. However PD166326 has some activity againstBaF3p210^(T315I) cells and inhibits their growth with IC₅₀ of 150 nM.This activity is related to Bcr-abl driven growth since growthinhibition of non-Bcr-abl driven cell types requires 5-15 fold higherconcentrations. Since PD166326 is a potent inhibitor of src kinases, andsince the src kinases Hck and Lyn mediate some of the transformingactivities of Bcr-abl, it is possible that PD166326 inhibits the growthof BaF3p210^(T315I) cells through the inhibition of Hck and Lyn.However, seemingly inconsistent with this hypothesis, we fail to seeinhibition of Hck Y⁴¹⁶ phosphorylation in these cells at growthinhibitory concentrations. However this does not disprove the hypothesisdue to limitations in assaying Hck activity in vivo. If PD166326 bindswith and inhibits the active Y⁴¹⁶ phosphorylated conformation of Hck,then this catalytically inactive drug-Hck complex may remain stably inthis phosphorylated conformation and phospho-Y⁴¹⁶ Hck antibodies will beunable to demonstrate the in vivo inhibition of Hck catalytic function.In vitro kinase assays do not help in this regard either, since duringthe process of cell lysis and immunoprecipitation, the Hck-PD166326interaction is lost. Therefore, in BaF3 p210^(T315I) cells, whereBcr-abl activity is resistant to PD166326, inhibition of Hck activitymay be responsible for the observed growth inhibitory effects atIC₅₀=150 nM despite persistent phosphorylation of Hck at these doses. Inaddition, although Y⁴¹⁶ is a site of auto-phosphorylation in srckinases, it may also be a substrate for phosphorylation by otherkinases. In fact in our experiments Hck Y⁴¹⁶ phosphorylation statusparallels Bcr-abl activity which suggests that Hck Y⁴¹⁶ may also be asubstrate for Bcr-abl. Although the activity of PD166326 against srckinases would suggest that it inhibits BaF3 p210^(T315I) cells through asrc family member, these experiments do not rule out the possibilitythat this cellular sensitivity is mediated through the inhibition ofother, yet unknown, kinases.

The structural basis for the STI571 resistance of the E255K mutatedBcr-abl is less clear since the functional significance of this residueis currently unknown. Interestingly this mutation confers littleresistance to PD166326. PD166326 shows no loss of activity againstBcr-abl^(E255K) autophosphorylation in vivo and only 2.5 fold lessactivity against the growth of BaF3Bcr-abl^(E255K) cells compared withwild type Bcr-abl controls. The cellular IC₅₀ of PD166326 againstBaF3Bcr-abl^(E255K) cells (15 nM) is much lower than its activity innon-Bcr-abl driven cell types (0.8-2 uM), and much greater than theactivity of STI571 against this mutant. If the basis for Bcr-abl E255Kresistance to STI571 is destabilization of the inactive conformation,and if PD166326 in fact binds to the active conformation, then thiswould explain why PD166326 is effective in inhibiting Bcr-abl^(E255K).However validation of these hypotheses requires crystal structure datato better define the function of the Glu²⁵⁵ residue and the binding ofPD166326 to Bcr-abl.

Tables

Tables 1A-1E identify typical MARS. The data are from analysis ofpatients, with an average of 10 clones sequenced per patient. Thesetables identify subgroups of mutations that are more likely to besignificant because they occur in more than one patient or they aredominant (defined as being detected in at least 2 of 10 clones in thesame patient). The observation that these mutants are showing up socommonly provides further evidence that these mutations will turn out tobe clinically significant.

TABLE IA Residues Mutated in Individuals Treated with STI-571 D233,T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259,K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282,F283, A288, M290, K291, E292, I293, P296, L298, V299, Q300, G303, V304,C305, T306, F311, I314, T315, E316, F317, M318, Y320, G321, D325, Y326,L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, I347, A350,M351, E352, E355, K357, M359, N358, F359, I360, L364, E373, N374, K378,V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, T406.

TABLE IB Typical Mutations at a Glance D233H, T243S, M244V, G249D,G250E, G251S, Q252H, Y253F, Y253H, E255K, E255V, V256L, Y257F, Y257R,F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R,D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G,I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S,C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C,Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G,L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K,E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D,K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G,H396K, A399G, P402T, T406A.

TABLE IC Mutations Occurring in More Than One Patient Q252H, E255K,K264R, F283S, M290T, P296S, V304D, T315I, R328K, M343T, M343V, A344T,M351T, K357E, M359V, I360T.

TABLE ID Dominant Mutations or Mutations with Frequencies Greater ThanOne Clone/Patient G250E, Q252H, Y253F, Y253H, E255K, V270A, V304D,T315I, F317L, M343T, M351T, E355G, M359V, I360K, V379I, F382L, H396K.

TABLE IE Mutations Occurring in More Than One Patient With a DominantClone or at Least Greater Than One Clone Occurrence Q252H, E255K, V304D,T315I, F317L, R328K, F359V, M351T.

TABLE IF Attached Mutations Y257F, Y274R, D276N, E282G, M290T, I293T,P296S, L298M, L298P, V304D, T315I, F317L, G321E, Q333L, A337V, V339G,M343T, M351T, E352A, I360T, E373K, V379I, D381G, F382L, T392A.

TABLE II GenBank accession number M14752 (SEQ ID NO: 1)MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDFEPQGLSEAARWNSKENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAEKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERIASGAITKGVVLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDIVQR

TABLE III Sensitivity of STI571-resistant BCR-ABL-transtormed cells togeldanamycin and 17-AAG. MEAN IC₅₀ ± S.D. (μM) CELL LINE GA 17-AAGBa/F3 + IL-3 27.3 ± 14.1 12.4 ± 0.3  Ba/F3 P210 WT 4.9 ± 1.6 5.2 ± 2.4Ba/F3 P210 T315I 1.8 ± 2.1 (P = 0.03)* 2.3 ± 0.4 (P = 0.04) Ba/F3 P210E255K 2.6 ± 2.4 (P = 0.05)  1.0 ± 0.2 (P = 0.01) Representative datafrom at least two independent experiments performed in duplicate; IC₅₀,concentration of inhibitor required to reduce the number of viable cellsby 50%; *Differences in the mean IC₅₀ values between WT and mutant P210Ba/F3 cells were analyzed with the unpaired Student's t-test; two-tailedP values are shown.

TABLE IV Detailed summary of Bcr-Abl kinase domain mutations by diseasecategory. Duration of Treatment # of independent with imatinib at clonescontaining Patient No. time of analysis Mutation(s) mutation  1 (MBC)4.5 months G250E (7/10) H396R (2/10)  2 (MBC) 13 months T315I (10/10)  3 (MBC) 8 months none N/A  4 (MBC) 3.5 months M351T (5/10)  5 (MBC) 1month Q252H (5/10)  6 (MBC) 13 months M351T (8/10)  7 (MBC) 13 monthsM351T (6/10)  8 (MBC) 1 month V304D (2/10)  9 (MBC) 5 months E255K(4/10) Y253H (2/10) 10 (MBC) 7 months E355G (5/10) F317L (2/10) 11 (MBC)6 months G250E (8/10) 12 (MBC) 3 months Y253F (3/10) E255K (2/10) M351T(2/10) H396R (2/10) 13 (MBC) 1.5 months M351T (3/10) T315I (2/10) Y253H(2/10) E255K (2/10) 14 (MBC) 3 months Y253F (2/10) E255K (2/10) T315I(2/10) 15 (MBC) 3 months E255K (2/10) 16 (MBC) 2 months E255K (2/10)Q252H (2/10) 17 (MBC) 4 months F359V (8/10) 18 (LBC) 1 month M351T(3/10) E255K (2/10) T315I (2/10) Y253F (2/10) 19 (LBC) T315I (5/10)E255K (2/10) Q252R (2/10) 20 (LBC) T315I (5/10) 21 (LBC) T315I (6/10) 22(LBC) none 23 (CPNCR) V379I (7/10) 24 (CPNCR) F317L (6/10) 25 (CPNCR)E255K (7/10) 26 (CPNCR) F359V (10/10)  27-35 none 36 (R-MBC) T315I(2/10) M343T (2/10) F382L (2/10) 37 (R-MBC) none 38 (R-MBC) none 39(R-MBC) none MBC denotes relapsed myeloid blast crisis despite STI-571.LBC denotes relapsed lymphoid blast crisis. CP denotes chronic phasewith no cytogenetic response. R-MBC denotes pre-STI-571 sample frommyeloid blast crisis patients whose disease was subsequently refractory.

Throughout this application, various publications are referenced. Thedisclosures of these publications are hereby incorporated by referenceherein in their entireties. The present invention is not to be limitedin scope by the embodiments disclosed herein, which are intended assingle illustrations of individual aspects of the invention, and anythat are functionally equivalent are within the scope of the invention.Various modifications to the models and methods of the invention, inaddition to those described herein, will become apparent to thoseskilled in the art from the foregoing description and teachings, and aresimilarly intended to fall within the scope of the invention. Suchmodifications or other embodiments can be practiced without departingfrom the true scope and spirit of the invention.

1. A method of identifying an amino acid substitution in at least oneBcr-Abl polypeptide expressed in a human cancer cell from an individualselected for treatment with a tyrosine kinase inhibitor, the methodcomprising determining the polypeptide sequence of at least one Bcr-Ablpolypeptide expressed by the human cancer cell and comparing thepolypeptide sequence of the Bcr-Abl polypeptide expressed by the humancancer cell to the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1 sothat an amino acid substitution in the Bcr-Abl polypeptide expressed bythe human cancer cell can be identified.
 2. The method of claim 1,wherein the amino acid substitution occurs in a region of the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1 comprising residue D233through residue T406.
 3. The method of claim 2, wherein the amino acidsubstitution occurs in the P-loop (residue G249 through residue V256 ofthe Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1), helix C(residue E279 through residue I293 of the Bcr-Abl polypeptide sequenceshown in SEQ ID NO: 1), the catalytic domain (residue H361 throughresidue R367 of the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1)or the activation loop (residue A380 through residue P402 of the Bcr-Ablpolypeptide sequence shown in SEQ ID NO: 1).
 4. The method of claim 2,wherein the amino acid substitution occurs at residue D233, T243, M244,K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263,K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288,M290, K291, E292, I293, P296, L298, V299, Q300, G303, V304, C305, T306,F311, I314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328,E329, Q333, E334, A337, V339, L342, M343, A344, I347, A350, M351, E352,E355, K357, N358, F359, I360, L364, E373, N374, K378, V379, A380, D381,F382, T389, T392, T394, A395, H396, A399, P402, or T406.
 5. The methodof claim 4, wherein the amino acid substitution is D233H, T243S, M244V,G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R,F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R,D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G,I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S,C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C,Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G,L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K,E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D,K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G,H396K, A399G, P402T or T406A.
 6. The method of claim 4, wherein theamino acid substitution occurs at residue G250, Q252, E255, K264, V270,F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35,K357, I360, V379 or H396.
 7. The method of claim 4, wherein the aminoacid substitution occurs at residue G250, Q252, Y253, E255, T315, F317,M351 or E355.
 8. The method of claim 4, wherein the amino acidsubstitution does not occur at residue E255.
 9. The method of claim 1,wherein the kinase inhibitor is a 2-phenylaminopyrimidine.
 10. Themethod of claim 9, wherein the amino acid substitution confersresistance to inhibition of tyrosine kinase activity by STI-571.
 11. Themethod of claim 1, wherein the polypeptide sequence of at least oneBcr-Abl polypeptide expressed by the human cancer cell is determined bydetermining the nucleotide sequence of a polynucleotide expressed by thehuman cancer cell that encodes the Bcr-Abl polypeptide.
 12. The methodof claim 11, wherein the Bcr-Abl polynucleotide expressed by the humancancer cell is isolated by the polymerase chain reaction.
 13. A methodof identifying a mutation in a Bcr-Abl polynucleotide in a mammaliancell wherein the mutation in a Bcr-Abl polynucleotide is associated withresistance to inhibition of Bcr-Abl tyrosine kinase activity by a2-phenylaminopyrimidine, the method comprising determining the sequenceof at least one Bcr-Abl polynucleotide expressed by the mammalian celland comparing the sequence of the Bcr-Abl polynucleotide to the Bcr-Ablpolynucleotide sequence encoding the polypeptide sequence shown in SEQID NO: 1, wherein the mutation in the Bcr-Abl polynucleotide comprisesan alteration at amino acid residue position: D233, T243, M244, K245,G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264,S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290,K291, E292, I293, P296, L298, V299, Q300, G303, V304, C305, T306, F311,I314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329,Q333, E334, A337, V339, L342, M343, A344, I347, A350, M351, E352, E355,K357, N358, F359, I360, L364, E373, N374, K378, V379, A380, D381, F382,T389, T392, T394, A395, H396, A399, P402, or T406 of the polypeptidesequence shown in SEQ ID NO:
 1. 14. The method of claim 13, wherein themammalian cell is a human cancer cell.
 15. The method of claim 14,wherein the human cancer cell is a chronic myeloid leukemia cell. 16.The method of claim 14, wherein the human cancer cell is obtained froman individual treated with STI-571.
 17. The method of claim 13, whereinthe substitution mutation occurs at residue G250, Q252, E255, K264,V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T,E35, K357, I360, V379 or H396.
 18. The method of claim 17, wherein thesubstitution mutation does not occur at residue E255.
 19. The method ofclaim 13, wherein the 2-phenylaminopyrimidine is STI-571.
 20. The methodof claim 19, wherein the amino acid substitution in the Bcr-Ablpolypeptide expressed in human cancer cell confers resistance toinhibition of tyrosine kinase activity by STI-571.
 21. The method ofclaim 13, wherein the polypeptide sequence of at least one Bcr-Ablpolypeptide expressed by the human cancer cell is determined bysequencing a polynucleotide expressed by the human cancer cell thatencodes the Bcr-Abl polypeptide, and wherein the Bcr-Abl polynucleotideexpressed by the human cancer cell is isolated by the polymerase chainreaction.
 22. A method of identifying a mutant Abelson protein tyrosinekinase expressed by a mammalian cancer cell, the method comprising: (a)determining a nucleotide sequence of a portion of a polynucleotideencoding the kinase domain of the Abelson protein tyrosine kinaseexpressed by the cell; and (b) comparing the nucleotide sequence sodetermined to that of the wild type sequence of the Abelson proteintyrosine kinase to identify the presence of a amino acid substitution inthe mutant Abelson protein tyrosine kinase, wherein any amino acidsubstitution so identified has the characteristics of occurring at aamino acid residue located within the polypeptide sequence of theAbelson protein tyrosine kinase at the same relative position as anamino acid substitution in the C-Abl protein kinase shown in SEQ ID NO:1 that has been identified as being associated with a resistance to aninhibition of tyrosine kinase activity by a 2-phenylaminopyrimidine, ascan be determined using the homology parameters of a WU-BLAST-2analysis.
 23. The method of claim 22, wherein the cell expressing themutant Abelson protein tyrosine kinase is found in a population ofmammalian cancer cells that are observed to exhibit a resistance to aninhibition of tyrosine kinase activity after exposure to a2-phenylaminopyrimidine.
 24. The method of claim 22, wherein themammalian cancer cell is a human cancer cell obtained from an individualselected for treatment with a tyrosine kinase inhibitor comprising a2-phenylaminopyrimidine.
 25. The method of claim 22, wherein themutation in the C-Abl protein kinase shown in SEQ ID NO: 1 that has beenidentified as being associated with a resistance to an inhibition oftyrosine kinase activity by a 2-phenylaminopyrimidine occurs at the samerelative position as amino acid residue D233, T243, M244, K245, G249,G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265,V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291,E292, I293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, I314,T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333,E334, A337, V339, L342, M343, A344, I347, A350, M351, E352, E355, K357,N358, F359, I360, L364, E373, N374, K378, V379, A380, D381, F382, T389,T392, T394, A395, H396, A399, P402, or T406.
 26. The method of claim 25wherein the amino acid substitution occurs at the same relative positionas amino acid residue G250, Q252, E255, K264, V270, F283, M290, P296,V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, I360, V379or H396.
 27. The method of claim 22 wherein the amino acid substitutionconfers resistance to inhibition of tyrosine kinase activity by ST-571.28. The method of claim 22, wherein the mutant Abelson tyrosine kinaseexpressed by the cell is a mutant c-Abl Bcr-Abl PDGFR, c-kit, TEL-Abl orTEL-PDGFR.
 29. The method of claim 22, further comprising repeatingsteps (a)-(b) another mammalian cancer cell obtained from a differentindividual; and (c) cataloging the mutations found in the mutant Abelsonprotein tyrosine kinases present in the mammalian cancer cells.
 30. Amethod of identifying a compound which specifically binds a mutantBcr-Abl polypeptide; wherein the Bcr-Abl polypeptide comprises an aminoacid substitution that occurs in a region of the Bcr-Abl polypeptidesequence shown in SEQ ID NO: 1 comprising residue D233 through residueT406, the method comprising the steps of: contacting the mutant Bcr-Ablpolypeptide with a test compound under conditions favorable to binding;and determining whether the test compound specifically binds to themutant Bcr-Abl polypeptide such that a compound which binds to themutant Bcr-Abl polypeptide can be identified.
 31. The method of claim30, wherein the amino acid substitution occurs at residue D233, T243,M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262,D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283,A288, M290, K291, E292, I293, P296, L298, V299, Q300, G303, V304, C305,T306, F311, I314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327,R328, E329, Q333, E334, A337, V339, L342, M343, A344, I347, A350, M351,E352, E355, K357, N358, F359, I360, L364, E373, N374, K378, V379, A380,D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406.
 32. Themethod of claim 31, wherein the amino acid substitution is D233H, T243S,M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F,Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C,Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R,E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D,C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T,Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V,V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A,E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K,N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A,A395G, H396K, A399G, P402T or T406A.
 33. The method of claim 30, whereinthe amino acid substitution occurs at residue G250, Q252, E255, K264,V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T,E35, K357, I360, V379 or H396.
 34. The method of claim 30, wherein theamino acid substitution occurs at residue G250, Q252, Y253, E255, T315,F317, M351 or E355.
 35. The method of claim 30, wherein the amino acidsubstitution does not occur at residue E255.
 36. The method of claim 30,wherein the compound is a 2-phenylaminopyrimidine.
 37. The method ofclaim 30, further comprising determining whether the test compoundinhibits the tyrosine kinase activity of the mutant Bcr-Abl polypeptidecomprising the steps of: transfecting mammalian cells with a constructencoding the mutant Bcr-Abl polypeptide; contacting the mammalian cellswith the test compound; and monitoring the mammalian cells for thetyrosine kinase activity of the mutant Bcr-Abl polypeptide, wherein aninhibition in tyrosine kinase activity in the presence of the testcompound as compared to the absence of the test compound identifies thetest compound as an inhibitor of the mutant Bcr-Abl polypeptide.
 38. Themethod of claim 37, wherein the amino acid substitution occurs atresidue T315.
 39. The method of claim 37, wherein the tyrosine kinaseactivity of the mutant Bcr-Abl polypeptide is measured by examining thephosphotyrosine content of Crkl.
 40. A method of determining whether atest compound inhibits the tyrosine kinase activity of a mutant Bcr-Ablpolypeptide, wherein the Bcr-Abl polypeptide comprises an amino acidsubstitution that occurs in a region of the Bcr-Abl polypeptide sequenceshown in SEQ ID NO: 1 comprising residue D233 through residue T406, themethod comprising the steps of: transfecting mammalian cells with aconstruct encoding the mutant Bcr-Abl polypeptide so that the mutantBcr-Abl polypeptide is expressed by the mammalian cells; contacting themammalian cells with the test compound; and monitoring the mammaliancells for the tyrosine kinase activity of the mutant Bcr-Ablpolypeptide, wherein an inhibition in tyrosine kinase activity in thepresence of the test compound as compared to the absence of the testcompound identifies the test compound as an inhibitor of the mutantBcr-Abl polypeptide.
 41. The method of claim 40, wherein the tyrosinekinase activity of the mutant Bcr-Abl polypeptide is measured byexamining the phosphotyrosine content of Crkl.
 42. The method of claim40, wherein the tyrosine kinase activity of the mutant Bcr-Ablpolypeptide is measured via Western blot analysis using ananti-phosphotyrosine antibody to examine the phosphotyrosine content oflysates of the mammalian cells.
 43. The method of claim 40, wherein themammalian cells are 293-T cells.
 44. The method of claim 40, wherein theamino acid substitution occurs at residue D233, T243, M244, K245, G249,G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265,V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291,E292, I293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, I314,T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333,E334, A337, V339, L342, M343, A344, I347, A350, M351, E352, E355, K357,N358, F359, I360, L364, E373, N374, K378, V379, A380, D381, F382, T389,T392, T394, A395, H396, A399, P402, or T406.
 45. The method of claim 44,wherein the amino acid substitution is D233H, T243S, M244V, G249D,G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R, F259S,K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N,T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T,P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S, C305Y,T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H,G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E,M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G,K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R,V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K,A399G, P402T or T406A.
 46. The method of claim 40, wherein the aminoacid substitution occurs at residue G250, Q252, E255, K264, V270, F283,M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357,I360, V379 or H396.
 47. The method of claim 40, wherein the amino acidsubstitution occurs at residue G250, Q252, Y253, E255, T315, F317, M351or E355.
 48. The method of claim 40, wherein the amino acid substitutiondoes not occur at residue E255.
 49. The method of claim 40, wherein thecompound is a 2-phenylaminopyrimidine.
 50. The method of claim 40,wherein the compound is a pyrido[2,3-d]pyrimidine.